B-homoestra-1,3,5(10)-trienes as modulators of tubulin polymerization

ABSTRACT

The invention provides B-ring expanded estra-1,3,5(10)-triene compounds of general formula (1) which modulate the polymerization of tubulin and/or the depolymerization of microtubules. The compounds have anti-angiogenic and anti-tumor activity. The invention also provides methods of preparing the compounds, and methods of using the compounds for the treatment of cancer or other mammalian diseases characterized by undesirable angiogenesis. The compounds of the invention are also expected to have utility as research tools.

This application is a 371 of PCT/US00/28273 filed Oct. 12, 2000, whichclaims benefit of U.S. Provisional Application No. 60/161,533 filed Oct.26,1999.

FIELD OF THE INVENTION

The present invention relates to the general field of steroid chemistry,particularly to estrone derivatives, and more particularly to B-ringexpanded estra-1,3,5(10)-triene compounds. The invention also relates tothe field of anti-mitotic, anti-tumor, and anti-angiogenic therapeutics,particularly to the field of therapeutics that function by modulation ofthe polymerization of tubulin and/or the depolymerization ofmicrotubules.

BACKGROUND OF THE INVENTION

1 Tubulin polymerization and Microtubule Assembly and Disassembly.

Cell mitosis is a multi-step process that includes chromosomereplication and cell division. It is characterized by the intracellularmovement and segregation of organelles, including mitotic spindles andthe replicated chromosomes. Organelle movement and segregation aredependent upon the polymerization of the heterodimeric cell proteintubulin into structures called microtubules. Successful cell division istherefore dependent upon the proper polymerization of tubulin, and alsoupon the proper functioning and subsequent disassembly of the resultingmicrotubules.

Tubulin and microtubules are sensitive to a variety of antimitoticdrugs. For example, colchicine and nocadazole are anti-mitotic drugsthat bind tubulin and inhibit tubulin polymerization, preventingmicrotubule formation. As such they have anti-tumor activity. Incontrast, the anti-cancer drug TAXOL™ binds to and stabilizes themicrotubules, inhibiting depolymerization and thereby interfering withthe later stages of mitosis. Thus, compounds that inhibit either thepolymerization or depolymerization of tubulin are potential antitumoragents. For reviews, see E. Hamel, Med. Res. Rev. 16:207-231 (1996) andL. Wang et al., Cancer Chemother Pharmacol, 44:355-361 (1999).

Three major pharmacological sites are present on tubulin: the colchicinesite, the vinca alkaloid domain, and the “taxoid” site. The latter siteis fully developed only in tubulin polymers with a well-definedprotofilament substructure. Parness and Horwitz, J Cell. Biol.91:479-487 (1981); Takoudju et al., FEBS Lett 227:96-98 (1988).

2. Non-steroidal Modulators of Tubulin Polyimerization.

Colchicine and nocadazole are anti-mitotic drugs that bind tubulin andinhibit tubulin polymerization. When used alone or in combination withother therapeutic drugs, colchicine in particular may be used to treatcancer. See for example PCT application WO 93/03729, and Japanese patent03240726 (1991). Allocolchicines, with a 7-membered B ring but6-membered C ring, have been reported, some of which are more activethan the corresponding colchicines. Ionio, Heterocycles 22:2207-2211(1984); Kang et al. J Biol. Chem. 265:10255-10259(1990)).

There are several cytotoxic vinca alkaloids that operate by themechanism of inhibition of tubulin polymerization. P. Verdier-Pinard etal., Biochem. Pharmacol. 58:959-971 (1999). In particular, the vincaalkaloid vinorelbine (NAVELBINE™) is a potent inhibitor of tubulinpolymerization that is currently approved for certain solid tumors.(Piccart, Cancer Treat. Rev., 23:S59 (1997). Cryptophycin is an evenmore potent inhibitor that is currently under investigation. D. Panda,Biochemistry, 36:12948 (1997).

Paclitaxel (TAXOL™) and docetaxel (TAXOTERE™) are examples of the taxaneclass of antimitotics, which bind to microtubules much more stronglythan they do to individual tubulin molecules. They have the effect ofaccelerating tubulin polymerization, and stabilizing the microtubulesagainst disassembly, which prevents successful completion of the mitoticprocess. For reviews, see Rowinsky, Ann. Rev. Med., 48:353 (1997),deFurla, Phytomedicine 4:273 (1997), and Balasubramanian et al., Ann.Reports Med. Chem., 33:151-162. These compounds are moderately effectiveagainst certain solid tumors. A combination of paclitaxel withvinorelbine has recently been approved by the F.D.A.

Compounds with similar biological effects to those of paclitaxel anddocetaxel include discodermolide, eleutherobin, sarcodictyin, and theepothilones. These compounds are in various stages of study and/ordevelopment as anti-tumor agents.

3. Steroidal Modulators of Tubulin Polymerization.

Among antimitotic agents that appear to bind at the colchicine site aresynthetic analogs of estradiol, such as diethylstilbestrol andestramustine, and the major endogenous metabolite of estradiol,2-methoxyestradiol (“2ME”). See for example R. D'Amato et al., Proc NatlAcad Sci USA, 91:3964-3968 (1994); M. Lottering et al., Cancer Res.52:5926-5923 (1992); L. Spicer and J. Hammond, Mol. and Cell. Endo.64:119-126 (1989); S. Rao and Engelberg, J Exp. Cell Res. 48:71-81(1967).

2-Methoxyestradiol (2ME) is a naturally occurring mammalian metaboliteof estradiol; it has very low affinity for the estrogen receptor. H.Breuer and R. Knuppen, Naturwissenschafte 12:280-281 (1960); H. Gelbkeand R. Knuppen, Steroid Biochem., 7:457-463 (1976). Interest in 2ME hasbeen stimulated by its cytotoxicity in cancer cell cultures, which ischaracterized by uneven chromosome distribution, faulty spindleformation, inhibition of DNA synthesis and mitosis, and an increase inthe number of abnormal metaphases. J. Seegers et al., J Steroid Biochem.32:797-809 (1989); M. Cushman et al,. J Med. Chem. 38:2041-2049 (1995).

2ME has been shown to bind to the colchicine binding site of tubulin,resulting in inhibition of tubulin polymerization and/or formation ofpolymer with altered stability properties and morphology. Hamel et al.,Biochemistry 35:1304-1310 (1996). Recent in vitro and in vivo resultshave shown that 2ME inhibits angiogenesis and tumor growth. Fotsis etal., Nature 368:237-239 (1994); Klauber et al., Cancer Res. 57:81-86(1997).

Efforts have been made to investigate the structure-activityrelationships of 2ME and its analogues, in an effort to design morepotent anticancer agents. Most work has been directed at modificationsin the steroid A ring, which is presumed to be analogous to thecolchicine tropolonic C ring. Among the compounds reported,2-ethoxyestradiol (2EE) has greater inhibitory effects on tubulinpolymerization and is 10-fold more cytotoxic than 2ME. Some6-substituted 2-ethoxyestradiols were synthesized and also demonstratedpromising biological activities. Estradiol analogs bearing acetyl groupsat positions C-2 and/or C-17 have also been evaluated for their effectson tubulin polymerization, but had minimal or no effect on tubulinpolymerization. H.-M. He and M. Cushman, Bioorg. Med. Chem. Lett.4:1725-1728 1994); M. Cushman et al., J. Med. Chem. 38:2041-2049 (1995);M. Cushman et al., J. Med. Chem. 40:2323-2334 (1997).

Miller et al., J Med. Chem. 40:3836-3841 (1997) disclosed 7-memberedtropolonic A ring analogs of 2ME, which were designed to enhance thesimilarity of the steroid A ring to the C ring of colchicine. They foundseveral of these A-homoestranes to be highly active inhibitors oftubulin assembly.

4. B-Homoestra-1 .3.5(10)-trienes.

B-Homoestra-1,3,5(10)-trienes are a little-known and very little-studiedclass of compounds. There have been few reported syntheses of suchcompounds, and even fewer biochemical or pharmaceutical studies. To thebest of the present inventors' knowledge, the five references discussedbelow constitute the known prior art in this area.

L. W. Rampy, in a thesis entitled “Total Synthesis of B-homoestrone andApproaches to Azaestrones” (1967, University of Michigan, Ann ArborMich.; Chemical Abstracts 68:96028; Diss. Abstr. B 1967, 28(6):2364)described the first total synthesis of racemic “B-homoestrone”(3-hydroxy-B-homoestra-1,3,5(10)-trien-17-one). Eleven intermediates andderivatives having 3-hydroxy and 3-methoxy groups were described. Noneof the compounds disclosed had more than a single hydroxy or methoxygroup on the A ring, and this group was always at the 3-position. Nobiological or biochemical activity of the compounds was disclosed,although “B-homoestrone” was prepared for the stated purpose ofinvestigating its potential as a modulator of plasma lipid levels.

E. E. Galantay, in French patent 1548354 (1968), disclosed, inter alia,the synthesis in racemic form of B-homoestra-1,3,5(10),8,14-pentaenes,having hydroxy, alkoxy, and acyloxy substituents at position 3. None ofthe disclosed compounds had more than a single substituent on the Aring, and the substituent was always at the 3-position. The compoundswere claimed to be useful for modulating estrogen-dependent conditions,e.g., endometriosis, dysmenorrhea, osteoporosis, and atherosclerosis,but no utility for the treatment of cancer was suggested.

E. E. Galantay and H. P. Weber, Experientia (1969) 25(6):571-572,described the total synthesis of racemic “B-homoestrone” and somederivatives, referring to them as “a hitherto unknown class ofcompounds.” These authors were evidently unaware of the previoussynthesis of B-homoestrone by L. Rampy. An X-ray crystal structure ofthe 3-methoxy derivative was reported. None of the compounds disclosedhad more than a single methoxy or hydroxy group on the A ring, and thissubstituent was always at the 3-position. No biological or biochemicalactivity of the compounds was disclosed.

E. Velarde, L. H. Knox, A. J. Cross, and P. Crabbe, Justus Liebigs Ann.Chem. (1971) 748:123-133, described7-fluoro-B-homoestra-1,3,5(10)-trienes, having hydroxy, methoxy, andacetoxy substituents at position 3. None of the disclosed compounds hadmore than a single substituent in the A ring, and all were at the3-position. No biological or biochemical activity of the compounds wasdisclosed.

M. B. Groen and F. J. Zeelen, Recl.: J. R. Neth. Chem. Soc. (1984)103(5):169-173, described the preparation of 1-methoxy and 3-methoxyB-homo-gona-1,3,5(10)-triene steroids, and subsequent transformations ofthe D ring to provide 3-methoxy-B-homoestra-1,3,5(10)-trien-17-one.However, none of the compounds described by Groen and Zelen had morethan a single methoxy group on the A ring. No biochemical,pharmacological, or biological activity of the compounds was disclosed.

In addition to the synthesis and characterization ofB-homoestra-1,3,5(10)-trienes described in the above references, thesecompounds fall into one of the many large classes of compoundsgenerically represented, but neither prepared nor characterized, in U.S.Pat. Nos. 5,504,074 and 5,661,143. These patents are incorporated hereinby reference in their entireties.

5. Summary

Although paclitaxel and docetaxel in particular have proved to bemedical and commercial successes, all of the active tubulin- ormicrotubule-modulating agents described above suffer from toxicityproblems, such as bone marrow suppression, hair loss, diarrhea, etc.They also tend to be specific for a limited class of tumor types. Thereremains a need for newer modulators of tubulin polymerization which mayprove to have improved selectivity and reduced side-effects.

BRIEF DESCRIPTION OF THE INVENTION

1. Definitions.

“Diseases characterized by undesirable cell mitosis” includes but is notlimited to excessive or abnormal proliferation of endothelial cells(e.g., psoriasis, atherosclerosis, endometriosis, hyperplasias), solidtumors and tumor metastasis, benign tumors, for example, hemangiomas,acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas,vascular malfunctions, abnormal wound healing, inflammatory and immunedisorders, Bechet's disease, gout or gouty arthritis.

“Diseases characterized by undesirable angiogenesis” includes but is notlimited to hematomas, and angiogenesis accompanying: solid tumors,rheumatoid arthritis, psoriasis, diabetic retinopathy and other ocularangiogenic diseases such as retinopathy of prematurity (retrolentalfibroplasic), macular degeneration, corneal graft rejection, neovascularglaucoma and Osler Weber syndrome. Normal but undesired processesinvolving angiogenesis, such as menstruation and the implantation of ablastula, are intended to fall within the meaning of the phrase as well.

The terms “alkyl” and “acyl” as used herein are intended to include bothstraight-chain and branched alkyl and acyl groups.

“MAP(s)” refers to microtubule-associated protein(s), as described in E.Hamel and C. Lin, Biochemistry, 23:4173-4184 (1984).

2. Description of the Invention

It has been discovered that certain compounds within the scope of theclaims below modulate tubulin polymerization and microtubule formationand/or depolymerization, and therefore are expected to be useful fortreating mammalian diseases characterized by undesired cell mitosisand/or undesired angiogenesis.

One object of this invention is to provide B-ring expandedestra-1,3-5(10)-triene derivatives of formula 1:

wherein the groups R¹, R², R³, A and B are as described furtherhereinbelow.

Another object of the invention is to provide pharmaceuticalcompositions comprising the compounds of the invention, and methods oftreating mammals in need of anti-tumor or anti-angiogenic therapy withthese compositions.

Yet another object of the invention is to provide methods of making2,3-disubstituted B-homoestra-1,3,5(10)-trienes of formula 1.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B illustrate methods of preparing certain representativecompounds of the invention, where the group A is CH2 or C—O.

FIG. 2 illustrates a method of preparing certain representativecompounds of the invention, where the group A is CHNHCOR

FIG. 3 illustrates a method of preparing certain representativecompounds of the invention, where the group A is NR⁴.

FIG. 4 is a series of turbidimetric curves showing enhancement ofglutamate-induced tubulin assembly (A: compounds 13 and 14; B: compound18) in the absence of MAPs.

FIG. 5 is a series of turbidimetric curves showing enhancement ofMAP-induced tubulin assembly by paclitaxel (curve 2), compound 13(curves 3-5), and compound 18 (curve 6), in the presence of MAPs.

FIG. 6 presents critical concentrations, obtained at 30° C., of tubulinwith paclitaxel and compound 13. A: MAPs -and GTP-dependent assemblywithout drug and in the presence of 10 μM paclitaxel or 10 μM 13. B:GTP-dependent, MAP-independent assembly with 10 μM paclitaxel or 10 μM13.

DETAILED DESCRIPTION OF THE INVENTION

In previous work, protected estradiol analogs bearing acetyl groups atpositions C2 and/or C17 have been evaluated for their effects on tubulinpolymerization. Invariably, these compounds had minimal or no effect ons polymerization (M. Cushman et al., J Med. Chem. 38:2041-2049 (1995);M. Cushman et al., J. Med. Chem. 40:2323-2334 (1997)). Nonetheless,compounds 13 and 18 of the present invention have been found tostimulate microtubule assembly in a manner qualitatively similar to thatobserved with the potent anticancer drug paclitaxel. Also surprisingly,compounds such as 11, 29, and 32 of the present invention have beenfound to inhibit tubulin polymerization. The presence of inhibitors andstimulators of tubulin polymerization within a single class of compoundsis a novel and unexpected observation.

Accordingly, this invention provides compounds having the followingstructure:

wherein A is CH₂, NR⁴, C═O, C═NOH, CHOH ,or CHNHCR⁵; B is CH₂ or C═O; R¹is H, methyl, ethyl, n-propyl, i-propyl, cyclopropyl, cyclopropylmethyl,1-propenyl, allyl, or vinyl, or other C₁ to C₆ alkyl, cycloalkyl, oralkenyl group; R² and R⁴ are independently H, methyl, ethyl, n-propyl,i-propyl, acetyl, propionyl, butyryl, cyclopropanecarbonyl, orisobutyryl, or other C₁ to C₆ alkyl, acyl, or cycloalkyl group; R³ is H,acetyl, propionyl, butyryl, cyclopropanecarbonyl, or isobutyryl, orother C₁ to C₆ acyl group; and R⁵ is H, methyl, ethyl, n-propyl,i-propyl, acetyl, propionyl, butyryl, cyclopropanecarbonyl, orisobutyryl, or other C₁ to C₆ alkyl, acyl, or cycloalkyl group

In preferred embodiments, A is C═O, CHOH, or CHNHCOR; B is CH₂; R¹ isselected from the group consisting of H, methyl, ethyl, 1-propenyl,n-propyl, and i-propyl; R² and R⁴ are selected from the group consistingof H, methyl, ethyl, n-propyl, i-propyl, acetyl, propionyl, butyryl, andisobutyryl; R³ is selected from the group consisting of H, acetyl,propionyl, butyryl, and isobutyryl; and R⁵ is selected from the groupconsisting of H, acetyl, propionyl, butyryl, and isobutyryl.

Another group of preferred compounds are those wherein A is NR⁴ and B isC═O.

More preferably, A is C═O or CHNHCOR; B is CH₂, R¹ is selected from thegroup consisting of methyl, ethyl, and 1-propenyl; R² is selected fromthe group consisting of H, acetyl, propionyl, butyryl, and isobutyryl;and R³ is selected from the group consisting of acetyl and propionyl.

Most preferably, A is C═O, B is CH₂, R¹ is selected from the groupconsisting of methyl, ethyl, and 1-propenyl; R² is selected from thegroup consisting of H and acetyl; and R³ is selected from the groupconsisting of acetyl and propionyl.

The compounds of this invention exhibit unexpected properties withrespect to their effect on polymerization of tubulin. In particular,depending upon the identities of the groups R², R³, A, and B, thecompounds either inhibit or enhance polymerization. For example, whereR¹ is ethyl, R² and R³ are H, and A and B are CH₂, the compound inhibitstubulin polymerization. Where R¹ is ethyl, R² and R³ are COCH₃, A isC═O, and B is CH₂, on the other hand, the compound stimulates tubulinpolymerization and stabilizes microtubules.

By the methods provided herein, and by obvious modifications thereto,the compounds of this invention may be prepared from the appropriatestarting materials. It will be appreciated that where A is CHOH orCHNHR⁴, the compounds may exist as either the α or β isomer at C-6, oras a mixture of the two compounds. Similarly, where B is CHOH, thecompounds may exist as either the α or β, isomer at C-7. It is intendedthat pure isomers, and mixtures thereof, are within the scope of theclaims. The compounds are presented merely by way of example, and arenot intended to limit the scope of the invention.

Another object of this invention is to provide methods of making thecompounds of the invention. The compounds may be prepared fromcommercially available steroids by the method outlined in Scheme 1 (FIG.1). Conversion of commercially available “β-estradiol”(estra-1,3,5(10)trien-3,17β-diol) to3,17-dialkoxyestra-1,3,5(9)trien-2-ols can be carried out by a publishedprocedure; see Z. Wang and M. Cushman, Synthetic Commun. 28:4431-4437(1998). Various protecting groups for the 3- and 17-hydroxyl groups,such as benzyl, silyl, and the like, may be employed as desired if theyare compatible with subsequent operations.

In the example provided,3,17β-bis(methoxymethoxy)estra-1,3,5(10)trien-2-ol is first treated withan alkylating agent, such as an alkyl halide or alkanesulfonate, in thepresence of an appropriate base. In the example provided, alkylationwith iodoethane gives 2. Deprotection of 2 under appropriate conditionsprovides the intermediate 3. Acylation of the two hydroxyl groups in 3provides 4, and oxidation at the benzylic 6-position with chromiumtrioxide in acetic acid then provides a 6-oxo intermediate such as 5.Use of other acylating agents, such as acyl chlorides and anhydrides, oruse of carboxylic acids in the presence of an activating agent, providesother embodiments with the desired identities of R² and R³. Where R² andR³ are desired to be different, a stepwise procedure will selectivelyintroduce R² first, at the less hindered and more acidic phenolicoxygen. If base-sensitive hydroxyl protecting groups such as acetate arepresent, they are removed, for example by treatment with ammonia or analkali metal hydroxide, and the hydroxyl groups are protected withbase-stable protecting groups such as t-butyldimethylsilyl ethers. Inthe present examples, the result is compound 7.

A key step in the method of synthesis of this invention is the B ringhomologation of an estra-1,3,5(10)-triene-6-one, which is not readilyaccomplished by the usual methods. It has been found thatestra-1,3,5(10)-triene-6-ones do not behave in a predictable fashionwhen traditional ring-expansion reactions are attempted. After manyunfruitful attempts, including for example attempted Tiffeneau-Demjanovrearrangements and diazomethane reactions, it has been found that twomore modem procedures, Taguchi's method and the TMSCHN₂/Et2O.BF3 method,could provide the desired one carbon ring-expansion products. However,even these methods generated unexpected regiochemical results, asdescribed below.

Taguchi's method involves a two-step sequence: nucleophilic addition ofdibromomethyllithium to a carbonyl group at C-6, followed by addition ofa strong base to generate a β-oxido carbenoid, which rearranges to aring-expanded lithium enolate. See Taguchi et al., J. Am. Chem. Soc.96:6510-6511 (1974); idem., Bull. Chem. Soc. Japan 50:1592-1595 (1977).

Treatment of compound 7 by Taguchi s method, using lithiumdiisopropylamide as base in the first step, gave the B-ring expandedproduct 8, in which aryl migration had occurred to leave the ketone atthe 7-position. The observed regioselectivity is entirely unexpected forthis type of compound, in view of the fact that when Taguchi andcoworkers used benzaldehyde to study this reaction, they obtainedexclusively the hydrogen migration product.

The problem was solved by relocating the carbonyl group from the7-position to the 6-position. The carbonyl group in 8 was reduced tomethylene with 80% yield by the method of Kabalka and Baker, J. Org.Chem. 40:1834-1835 (1975), involving reduction of tosylhydrazone 10 withcatecholborane followed by thermal decomposition. After acetylation ofthe two hydroxyl groups of 11, oxidation of 12 with chromium trioxide inacetic acid proceeded selectively at the 6-position to give3,17β-diacetate 13. Saponification then providedB-homo-3,17β-dihydroxy-2-ethoxyestra-1,3,5(10)-triene-6-one 14.

Alternatively, the ring enlargement of 6 directly to 14 may beaccomplished by using trimethylsilyldiazomethane and boron trifluorideetherate (TMSCHN₂/Et₂O.BF₃) by the method of Seto et al., TetrahedronLett., 40:2359-2362 (1999). Seto et al. homologated 6-oxo-steroids togive selectively B-homo-6-oxo-steroids in ca. 80% isolated yield.However, reaction of ketone 5 with TMSCHN₂ in the presence of Et₂O.BF₃at −20° C. followed by acid treatment gave an inseparable mixture ofB-ring homologated estradiol derivatives (13 and its 7-oxo isomer). TheNMR spectrum indicated the presence of an equimolar mixture of the twoketones, but all attempts to separate these two isomers wereunsuccessful. This problem was solved by removal of the acetyl groups,after which the B-homoestradiol compounds 14 (24%) and 9 (31%) could beisolated by column chromatography.

The reductive amination of compound 14, followed by acylation, is aneffective approach to the respective acetamides. The 6α-epimer 23 and6β-epimer 24 could be isolated by flash chromatography on silica gelusing a suitable solvent system with careful operation. The ratio of therespective isomers obtained from the reductive amination were found tobe 1 (α):2(β). Hydrolyses of 23 and 24 were accomplished with dilutesodium hydroxide solution in methanol, which removed the 3 and 17 acetylgroups to afford the corresponding 25 and 26, respectively.

Extensive NMR analysis of compounds 25 and 26 was performed in order toelucidate their structure, absolute configuration and conformation. Thestereochemistry at C-6 in 25 and 26 was deduced by NOESY experiment. Avery strong NOE between H-6 and H-9 was observed in the spectra of 26,which was absent in the spectra of 25. The assigned C-6 configurationswere confirmed by X-ray crystallography.

The mono-protected B-homoestra-1,3,5(10)-trien-3,17-diols bearing oneacyl group at the 17-position (e.g., 18) or at the 3-position (e.g., 19)are prepared by selective hydrolysis of a phenolic ester such as 13 orby selective acylation of the phenolic group of a 3,17-diol such as 14.Thus, mild basic hydrolysis of compound 13 with KHCO₃ in methanol at 65°C. for 1.5 hr provided the 17-βmonoacetate 18, while treatment of 14with 1-acetyl-1H-triazolo[4,5-b]pyridine and 1 N NaOH in THF providedthe monoacetate 19 in high yield. The 6-ketones of this invention, forexample 13, 14, and 18, can be transformed to oximes, for example 21, 21or methoximino compound 22, by treatment with the appropriatehydroxylamine or alkoxylamine. The 6,7-dehydro compound 16 was preparedby the treatment of compound 10 with methyllithium in anhydrous THF.

Compounds of this invention where A is NR can be prepared byrearrangement of a 6-oximinoestra-1,3,5-(10)-triene. A preferred methodis by base-catalyzed rearrangement of a 6-(O-arylsulfonyloximino)derivative, for example the rearrangement of a6-(O-toluenesulfonyloximino)derivative such as 28 catalyzed by basicalumina. The resulting B-ring lactam 29 may be reduced, alkylated,acylated, etc. by well-known methods, providing for example compoundssuch as 30-33.

It is anticipated that prodrug forms of the compounds of this inventionwill prove useful in certain circumstances, and such compounds areintended to fall within the scope of the invention. Prodrug forms mayhave advantages over the parent compounds of formula 1 in that they arebetter absorbed, better distributed, more slowly metabolized or cleared,etc. Prodrug forms may also have formulation advantages in terms ofcrystallinity or water solubility. For example, compounds of theinvention having one or more hydroxyl groups may be converted to estersor carbonates bearing one or more carboxyl, hydroxyl or amino groups,which are hydrolyzed at physiological pH values or are cleaved byendogenous esterases or lipases in vivo. See for example U.S. Pat. Nos.4,942,184, 4,960,790, 5,817,840, and 5,824,701 (all of which areincorporated herein by reference in their entirety), and referencestherein.

Another object of this invention is to provide a method of treating anindividual with cancer, or another disease characterized by undesirableangiogenesis, with compounds of formula 1. The method of the inventioncomprises administering to an individual a therapeutically effectiveamount of at least one compound of formula 1, or a prodrug thereof,which is sufficient to inhibit tumor growth.

The dose of the compound used in the treatment of such disease will varyin the usual way with the weight and metabolic health of the patient,and with the relative efficacy of the compound employed when usedagainst the type of tumor involved. The preferred initial dose for thegeneral patient population will be determined by routine dose-rangingstudies, as are conducted for example during clinical trials.Therapeutically effective doses for individual patients may bedetermined by titrating the amount of drug given to the individual toarrive at the desired therapeutic effect without incurring anunacceptable level of side effects, as is currently done with otherforms of chemotherapy.

For example, the compound 11 would be expected to be useful at dosageswhich are about 3 to 5 times those used for taxotere. A preferredinitial dose for this compound, accordingly, may be estimated to bebetween about 10 and 2000 mg/day for an adult human, more preferablybetween 100 and 1000 mg/day. The initial dose may be varied so as toobtain the optimum therapeutic effect in the patient, and may beprovided as a daily dose, in a divided dose regimen, or by continuousinfusion.

Administration of the compounds of this invention may be by any methodused for administering therapeutics, such as for example oral,parenteral, intravenous, intramuscular, subcutaneous, or rectaladministration.

This invention also provides pharmaceutical compositions useful forproviding anti-tumor activity, which comprise at least one compound ofthe invention. In addition to comprising at least one of the compoundsdescribed by formula 1 or a pro-drug thereof, the pharmaceuticalcomposition may also comprise additives such as preservatives,excipients, fillers, wetting agents, binders, disintegrants, buffers,and/or carriers. Suitable additives may be for example magnesium andcalcium carbonates, carboxymethylcellulose, starches, sugars, gums,magnesium or calcium stearate, coloring or flavoring agents, and thelike. There exists a wide variety of pharmaceutically acceptableadditives for pharmaceutical dosage forms, and selection of appropriateadditives is a routine matter for those skilled in art of pharmaceuticalformulation.

The compositions may be in the form of tablets, capsules, powders,granules, lozenges, suppositories, reconstitutable powders, or liquidpreparations such as oral or sterile parenteral solutions orsuspensions.

In order to obtain consistency of administration it is preferred that acomposition of the invention is in the form of a unit dose. Unit doseforms for oral administration may be tablets, capsules, and the like,and may contain conventional excipients such as binding agents, forexample syrup, acacia, gelatin, sorbitol, tragacanth, orpolyvinylpyrrolidone; and carriers or fillers, for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine. Additivesmay include disintegrants, for example starch, polyvinylpyrrolidone,sodium starch glycolate or microcrystalline cellulose; preservatives,and pharmaceutically acceptable wetting agents such as sodium laurylsulfate.

In addition to unit dose forms, multi-dosage forms are also contemplatedto be within the scope of the invention. Delayed-release compositions,for example those prepared by employing slow-release coatings,micro-encapsulation, and/or slowly-dissolving polymer carriers, willalso be apparent to those skilled in the art, and are contemplated to bewithin the scope of the invention. For example, the compounds of thisinvention may be incorporated into biodegradable polymers allowing forsustained release, the resulting compositions preferably being implantedwhere delivery is desired, for example, at the site of a tumor.Biodegradable polymers suitable for this embodiment are well-known inthe art, see for example Brem et al., J. Neurosurg. 74:441-446 (1991).The compounds of this invention may be also be incorporated into othersustained-release formulations, such as those employing coatedparticles. See for example U.S. Pat. No. 5,968,551 and referencestherein.

The solid oral compositions may be prepared by conventional methods ofblending, filling, tabletting or the like. Repeated blending operationsmay be used to distribute the active agent throughout those compositionsemploying large quantities of fillers. Such operations are conventionalin the art. The tablets may be coated according to methods well known innormal pharmaceutical practice, for example with an enteric coating.

Oral liquid preparations may be in the form of, for example, emulsions,syrups, or elixirs, or may be presented as a dry product forreconstitution with water or other suitable vehicle before use. Suchliquid preparations may contain conventional additives such assuspending agents, for example sorbitol syrup, methyl cellulose,gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminumstearate gel, and hydrogenated edible fats; emulsifying agents, forexample lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles(which may include edible oils), for example almond oil or fractionatedcoconut oil, oily esters such as esters of glycerin, propylene glycol,or ethyl alcohol; preservatives, for example methyl or propylp-hydroxybenzoate or sorbic acid; and if desired conventional flavoringor coloring agents.

For parenteral administration, which will be a preferred route ofadministration in the hospital or cancer clinic environment, fluid unitdosage forms are prepared utilizing the compound and a sterile vehicle.Depending on the concentration used, the compound can be eithersuspended or dissolved in the vehicle. In preparing solutions thecompound can be dissolved in water or saline for injection and filtersterilized before filling into a suitable vial or ampoule and sealing.Advantageously, additives such as a local anaesthetic, a preservativeand buffering agents can be dissolved in the vehicle. Suitable bufferingagents are, for example, phosphate and citrate salts. To enhance thestability, the composition can be frozen after filling into the vial andthe water removed under vacuum.

Parenteral suspensions are prepared in substantially the same manner,except that the compound is suspended in the vehicle instead or beingdissolved, and sterilization accordingly cannot readily be accomplishedby filtration. The compound can be sterilized by filtration of analcohol solution, or by other conventional means, for example byexposure to radiation before or after being suspended in the sterilevehicle. Advantageously, a surfactant or wetting agent is included inthe composition to facilitate uniform distribution of the compound andstability of the suspension.

All references cited in this disclosure are incorporated by referenceherein, in their entirety.

EXAMPLES

Melting points were determined in capillary tubes on a Mel-Tempapparatus and are uncorrected. ¹H NMR spectra were recorded on a VarianVXR-300S spectrometer using TMS as an internal standard; Cl mass spectrawere obtained on a Finnegan 4000 spectrometer; El mass spectra on aKratos MS50 spectrometer; IR spectra on a Perkin Elmer 1600 series FTIR.Microanalyses were performed at the Purdue Microanalysis Laboratory.

Flash column chromatography was carried out using Merck silica gel(230-400 mesh). Analytical thin-layer chromatography (TLC) was performedon prescored silica gel GF coated glass plates (Analtech; 2.5×10 cm with250 μM layer), and spots were visualized with UV light at 254 nm or with5% H₂SO₄ in ethanol. Most chemicals and solvents were analytical gradeand used without further purification. Commercial reagents werepurchased from Aldrich Chemical Company (Milwaukee, Wis.).

3,17β-Bis(methoxymethoxy)-2-ethoxyestra-1,3,5-triene (2). A solution of3,17β-bis(methoxymethoxy)estra-1,3,5-triene-2-ol (Z. Wang and Cushman,Synzthetic Commun. 28:4431-4437 (1998)) (11.3 g, 27.8 mmol) in anhydrousethanol (225 ml) containing anhydrous potassium carbonate (38.4 g, 280mmol) was stirred at room temperature under argon for 10 min. Iodoethane(43.7 g, 280 mmol) was introduced to the reaction mixture. The resultingmixture was stirred at gentle reflux. After 4 hr, another portion ofiodoethane (10.92 g, 70 mmol) was introduced and the reflux wascontinued for another 10 hr. The reaction mixture was cooled to roomtemperature and filtered. The solid was washed with ether (100 ml), andthe filtrates were combined and evaporated to dryness. Chromatography ofthe residue (silica gel 230-400 mesh, ethyl acetate:hexane 1:7 byvolume) gave the pure 2 (10.45 g, 92%) as a colorless oil: ¹H NMR(CDCl₃) δ6.85 (s, 2 H), 5.18 (s, 2 H), 4.66 (ABq, J=6.6 Hz, Δν=3.4 Hz, 2H), 4.08 (q, J=7.0 Hz, 2 H), 3.61 (t, J=8.5 Hz, 1 H), 3.52 (s, 3 H),3.38 (s, 3 H), 2.79 (m, 2 H), 2.30-1.20 (m, 13 H), 0.82 (s, 3 H); CIMS(isobutane) m/z (rel intensity) 405 (MH+, 39), 343 (100). Anal. Calcd.for C₂₄H₃₇O₅: C, 71.26; H, 8.97. Found: C, 71.48; H, 9.14.

2-Ethoxyestra-1,3,5(10)-triene-3.17β-diol (3). To a solution of 2 (6.0g, 14.8 mmol) in THF (100 ml) was added 6 N HCl (100 ml) at roomtemperature and the resulting solution was stirred at room temperaturefor 6 hr. The reaction mixture was poured into brine (200 ml) and theproducts were extracted with ethyl acetate (3×100 ml). The ethyl acetatelayers were washed with saturated sodium bicarbonate (100 ml) and brine(100 ml), combined and dried over sodium sulfate, and evaporated todryness. Chromatography of the residue (silica gel 230-400 mesh,methylene chloride:ethyl acetate 9:1 by volume) gave compound 3 (4.04 g,85%), which was crystallized from ethyl acetate/hexane to afford whitecrystals: mp 151-150° C. (lit.4 mp 154-155° C.); ¹H NMR (CDCl₃) δ6.95(s, 1 H), 6.65 (s, 1 H), 4.08 (qd, J=6.8 Hz, J=1.5 Hz, 2 H), 3.73 (t,J=8.4 Hz, 1 H), 2.73 (m, 2 H), 2.30-1.20 (m, 18 H), 0.81 (s,3 H).

3,17β-Diacetoxy-2-ethoxyestra-1,3,5(10)-triene (4). Acetic anhydride (20ml, 210 mmol) was added under nitrogen at room temperature to a solutionof 3 (3.89 g, 12.3 mmol) in anhydrous pyridine (34 ml). The resultingmixture was stirred at room temperature for 24 hr and then poured intoice/water mixture (200 ml). The product was extracted with ethyl acetate(3×100 ml). The organic layers were washed with water (100 ml), aqueoussodium bicarbonate (100 ml) and brine (2×100 ml), and dried over sodiumsulfate. The ethyl acetate layer, on evaporation under reduced pressure,gave compound 4 (4.82 g, 98%), which was crystallized from ethylacetate/hexane to afford white crystals: mp. 132-133° C. (lit.12 mp135-136° C.); ¹H NMR (CDCl₃) δ6.95 (s, 1 H), 6.75 (s, 1 H), 4.71 (t,J=8.5 Hz, 1 H), 4.09 (qd, J=6.9 Hz, J=1.5 Hz, 2 H), 2.73 (m, 2 H), 2.36(s, 3 H), 2.12-2.4 (m, 3 H), 2.10 (s, 3 H), 1.1-2.0 (m, 13 H), 0.85 (s,3 H).

3,17β-Diacetoxy-2-ethoxyestra-1,3,5(10)-triene-6-one (5). A solution ofchromium trioxide (5.14 g, 51.4 mmol) in 90% glacial acetic acid (50 ml)was added dropwise at 10-12° C. to a mechanically-stirred solution of 4(4.82 g, 12.03 mmol) in glacial acetic acid (77 ml). After the addition,the resulting mixture was stirred at 10-12° C. for 40 min. The mixturewas poured into ice-water mixture (400 ml) and the compounds wereextracted with ethyl acetate (300, 200, and 100 ml). The organic layerswere washed with brine (2×200 ml), solution of sodium bicarbonate (2×150ml) and brine (2×200 ml), dried over sodium sulfate and evaporated todryness. Chromatography of the residue on a silica gel column using 30%ethyl acetate in hexane gave the title compound 5 (3.9 g, 78%), whichwas crystallized from ethyl acetate/hexane to afford white crystals: mp193-194° C. (lit.12 mp 195° C.); IR (KBr) 1774, 1736, 1719 (C═O); ¹H NMR(CDCl₃) δ7.75 (s, 1 H), 6.95 (s, 1 H), 4.75 (t, J=8.5 Hz, 1 H), 4.19 (q,J=6.9 Hz, 2 H), 2.85-2.48 (m, 2 H), 2.45-1.1 (m, 19 H), 0.85 (s, 3 H).

3,17β-Dihydroxy-2-ethoxyestra-1,3,5(10)-trien-6-one (6). Under nitrogen,a 20% solution of potassium hydroxide in methanol (15 ml) was addeddropwise at room temperature to a suspension of compound 5 (3.0 g, 7.26mmol) in anhydrous methanol (60 ml). The resulting mixture was stirredat room temperature for 6 hr. The mixture was neutralized with 3 N HCland the solvent was removed under reduced pressure. The residue wasdiluted with water (100 ml) and extracted with ethyl acetate (3×100 ml).The combined organic layer was washed with brine (2×100 ml), and driedover sodium sulfate. Removal of the solvents provided a white solid 6(2.34 g, 98%), which was then crystallized from ethyl acetate/hexane toafford title compound 6 as white crystals: mp 197-199° C. (lit. 12 mp194-196° C.); ¹H NMR (CDCl₃) δ7.61 (s, 1 H, 4-aromatic CH), 7.20 (s, 1H, 1-aromatic CH), 4.44 (q, J=6.9 Hz, 2 H, 2-CH2-), 3.83 (t, J=8.5 Hz, 1H, 17α-H), 2.85-1.40 (m, 20H), 0.97 (s, 3 H, 18-CH3).

3,17β-Bis(t-butyldimethylsilyloxy)-2-ethoxyestra-1,3,5(10)-triene-6-one(7). Under argon, a solution of compound 6 (4.92 g, 14.9 mmol),imidazole (12.3 g, 178.4 mmol) and t-butyldimethylchlorosilane (i 3.5 g,89.6 mmol) in DMF (100 ml) was stirred overnight at room temperature.The reaction mixture was poured into ice/cold sodium bicarbonatesolution (250 ml) and the product was extracted with ethyl acetate (200ml, 150 ml, 100 ml). The organic layers were washed with water (200 ml)and brine (2×100 ml), dried over sodium sulfate and evaporated todryness. Crystallization of the residue from methanol gave compound 7(7.6 g, 91%), which was obtained as white crystals: mp 150-151° C.; ¹HNMR (CDCl₃) δ7.51 (s, 1 H), 6.78 (s, 1 H), 4.19 (q, J=6.9 Hz, 2 H), 3.65(t, J=8.5 Hz, 1 H), 2.85-2.48 (m, 2 H), 2.45-1.1 (m, 19 H), 0.98 (s, 9H), 0.86 (s, 9 H), 0.72 (s, 3 H), 0.15 (s, 6 H), 0.01 (s, 6 H); CIMS(isobutane) m/z (rel intensity) 559 (MH+, 100). Anal. Calcd forC₃₂H₅₄O₄Si₂: C, 68.76; H, 9.74. Found: C, 68.61; H, 9.90.B-Homo-3,17β-bis(t-butyldimethylsilyloxy)-2-ethoxyestra-1,3,5(10)-triene-7-one

(8) Under argon , a well-stirred solution of7 (6.38 g, 11.41 mmol) anddibromo methane (11.9 g, 68.5 mmol) in dry THF (190 ml) was cooled to−78° C. and then the solution was treated with lithium diisopropylamidemono(tetrahydrofuran) (30.4 ml, 1.5 M solution in cyclohexane, 45.6mmol) dropwise over a period of 1.5 hr. After stirring 3 hr at the samelow temperature, n-butyllithium (60 ml, 1.6 M solution in hexane, 96mmol) was added to the mixture over a period of 1.5 hr. The resultingred solution was stirred for another 1 hr at −78° C. and 10 min at 0°C., quenched by pouring into ice (150 g), and then extracted with ethylacetate (3×100 ml). The organic layers were washed with brine (3×100ml), combined and dried over sodium sulfate, and evaporated to dryness.Chromatography of the residue (silica gel 230-400 mesh, methylenechloride:hexane 7:3 by volume) recovered the starting material 7 (1.98g) and gave the title compound 8 as an oil (2.78 g, 61.6% based on theconsumption of 7). Even if large excess of reagents were used, about25-30% of starting material was recovered in most cases. IR (KBr, cm⁻¹)2955, 2930, 1709 (7-C═O), 1511; ¹H NMR (CDCl₃) δ6.78 (s, 1 H), 6.60 (s,1 H), 4.04 (qd, J=7.0 Hz, J 1.5 Hz, 2 H), 3.66 (t, J=8.5 Hz, 1 H,17α-H), 3.60 (d, J=20.1 Hz, 1 H), 3.28 (d, J=20.3, 1 H), 2.68 (dd,J=11.3 Hz, J=8.5 Hz, 1 H), 2.34 (m, 1 H), 2.2-1.20 (m, 16 H), 0.98 (s, 9H), 0.88 (s, 9 H), 0.78 (s, 3 H), 0.13 (s, 6 H), 0.05 (s, 6 H); CIMS(isobutane) m/z (rel intensity) 573 (MH+, 100). Anal. Calcd forC₃₃H₅₆O₄Si₂: C, 69.18; H, 9.85. Found: C, 68.96; H, 9.91.

B-Homo-3,170β-dihydroxy-2-ethoxyestra-1,3,5(10)-triene-7-one (9). Undernitrogen, a mixture of 8 (3.42 g, 5.97 mmol) and a 1.0 M solution oftetrabutylammonium fluoride in THF (50 ml, 50 mmol) was stirred at roomtemperature for 6 hr. The reaction mixture was poured into an ice-coldsodium bicarbonate solution (200 ml) and then extracted with ethylacetate (3×100 ml). The ethyl acetate was washed with brine (2×100 ml),dried over sodium sulfate, and evaporated to dryness. Chromatography ofthe residue (silica gel 230-400 mesh, ethyl acetate:hexane 1:1 byvolume) gave the compound 9 as white crystals (1.8 g, 87.4%): mp210-212° C.; IR (KBr, cm⁻¹) 3403 (br., OH), 2970, 2855, 1688, 1582,1510; ¹H NMR (CDCl₃+DMSO-d₆) 6.82 (s, 1 H), 6.69 (s, 1 H), 4.14 (q,J=7.0 Hz, 2 H), 3.69 (t, J=8.5 Hz, 1 H), 3.62 (d, J=20.1 Hz, 1H), 3.32(d, J=19.8, 1 H), 2.68 (dd, J=11.3 Hz, J=8.5 Hz, 1 H), 2.34-1.20 (m, 16H), 0.88 (s, 3 H, CH3); CIMS (isobutane) m/z (rel intensity) 345 (MH+,100). Anal. Calcd for C₂₁H₂₈O₄: C, 73.23; H, 8.19. Found: C, 73.15; H,7.97.

B-Homo-2-ethoxy-3,17β-dihydroxyestra-1,3,5(10)-triene-7-onetoluenesulfonyl-hydrazone (10). Under nitrogen and at room temperature,a mixture of compound 9 (1.73 g, 5.02 mmol) andp-toluenesulfonylhydrazine (4.67 g, 25.1 mmol) in methanol (100 ml) wasstirred for 24 hr. The resultant mixture was filtered and the solidwashed with cold methanol to afford tosylhydrazone 10 as white crystals(2.48 g, 96.5%). The analytical sample was recrystallized from methanol:mp 179-181° C.; IR (KBr, cm⁻¹) 3428 (br., OH, NH), 2922, 1624, 1594, 151; ¹H NMR (CDCl₃) δ7.88 (d, J=8.0 Hz, 2 H), 7.34 (d, J=8.0 Hz, 2 H), 7.33(s, 1 H, NH), 6.71 (s, 1 H), 6.68 (s, 1 H), 5.55 (s, 1 H, OH), 4.11 (q,J=7.0 Hz, 2 H), 3.58 (d, J=19.1 Hz, 2 H), 3.39 (s, 1 H, OH), 3.45 (t,J=8.5, 1 H), 3.15 (d, J=19.1 Hz, 1 H), 2.17-0.99 (m, 16 H), 0.78 (s, 3H, CH3); CIMS (isobutane) m/z (rel intensity) 360 (MH+, 100), 342 (51).Anal. Calcd. for C₂₁H₂₉O₄N.1/6H2O: C, 69.65; H, 8.16; N, 3.86. Found: C,69.81; H, 8.06; N, 3.89.

B-Homo-2-ethoxy-3.17β-estradiol (11). Under argon, a solution of 10(2.27 g, 4.43 mmol) in anhydrous chloroform (150 ml) was cooled to ⁰° C.and then catecholborane (44.3 ml, 1.0 M solution in THF, 44.3 mmol) wasadded dropwise at the same temperature. The resultant mixture wasstirred for 10 hr, after that sodium acetate trihydrate (12.06 g, 88.62mmol) was added in portions. The mixture was allowed to warm to roomtemperature over 30 min and then heated under reflux for 6 hr. Thereaction mixture was cooled to room temperature and filtered. The solidmaterial was washed with chloroform (100 ml) and the combined filtrateswere evaporated under reduced pressure to dryness. The remaining oil waspurified by chromatography on a silica gel column with ethylacetate:hexane 1:3 by volume. The product was crystallized from ethylacetate/hexane to give the title compound 11 as white crystals (1.17 g,80.1%): mp 86-88° C.; IR (KBr, cm⁻¹) 3490 (OH), 3259 (OH), 2925, 2861,1607, 1511; ¹ H NMR (CDCl₃+DMSO-d₆) δ6.81 (s, 1 H), 6.67 (s, 1 H), 5.46(s, 1 H, OH), 4.11 (dq, J=3.3 Hz, J=7.0 Hz, 2 H), 3.74 (t, J=8.2 Hz, 1H), 2.86 (d,t, J=14.6 Hz, J=7.3, 1H), 2.64-2.47 (m, 2 H), 2.14-1.22 (m,18 H) 0.83 (s, 3 H, CH₃); CIMS (isobutane) m/z (rel intensity) 330 (M+,100). Anal. Calcd for C₂₁H₃₀O₃: C, 76.33; H, 9.15. Found: C, 76.54; H,9.44.

B-Homo-3,17β-diacetoxy-2-ethoxyestra-1,3,5(10)-triene (12). Aceticanhydride (6 ml, 63 mmol) was added under argon at room temperature to asolution of diol 11 (1.14 g, 3.45 mmol) in anhydrous pyridine (12 ml).The resulting mixture was stirred at room temperature for 24 hr and thenpoured into ice/water mixture (100 g). The compound was extracted withethyl acetate (3×70 ml). The organic layers were washed with water (100ml), aqueous sodium bicarbonate (2×100 ml) and brine (2×100 ml), driedover sodium sulfate and evaporated to dryness. Chromatography of theresidue on silica gel (230-400 mesh) using ethyl acetate:hexane 1:1 byvolume gave the compound 12 (1.43 g, 100%), which was crystallized fromethyl acetate/hexane to afford white crystals: mp 98-100° C.; IR (KBr,cm⁻¹) 2928, 2870, 1768 (3-C═O), 1735 (17-C═O), 1510; ¹H NMR (CDCl₃)δ6.89 (s. 1 H), 6.75 (s, 1 H), 4.70 (t, J=8.7 Hz, 1 H, 17α-H), 4.06 (dq,J=3.3 Hz, J=7.0 Hz, 2 H), 2.89 (dt, J=14.6 Hz, J=7.3, 1 H), 2.64-2.52(m, 2 H), 2.29 (s, 3 H, COCH₃), 2.07 (s, 3 H, COCH₃), 2.23-1.26 (m, 18H), 0.88 (s, 3 H, CH₃); CIMS (isobutane) m/z (rel intensity) 415 (MH+,48), 355 (100). Anal. Calcd for C₂₅H₃₄O₅: C, 72.43; H, 8.27. Found: C,72.79; H, 8.49.

B-Homo-3,17β-diacetoxy-2-ethoxyestra-1,3,5(10)-triene-6-one (13). Asolution of chromium trioxide (1.34 g, 13.4 mmol) in 90% glacial aceticacid (13 ml) was added dropwise over a period of 20 min at 13-15° C. toa well-stirred solution of compound 12 (1.29 g, 3.11 mmol) in glacialacetic acid (35 ml), the resulting mixture was then stirred at 13-15° C.for 15 min. The mixture was poured into ice-water mixture (300 g) andextracted with ethyl acetate (3×200 ml). The combined organic layerswere washed with brine (2×100 ml), aqueous sodium bicarbonate (2×100 ml)and brine (2×100 ml), dried over sodium sulfate and evaporated todryness. Chromatography of the residue on a silica gel column using 20%ethyl acetate in hexane gave ketone 13 (925 mg, 69.3%), which wascrystallized from ethyl acetate/hexane to afford white crystals: mp178-180° C.; IR (KBr, cm⁻¹) 2935, 2865, 1774 (3-C═O), 1728 (17-C═O),1669 (6-C═O), 1500; ¹H NMR (CDCl₃) δ7.36 (s, 1 H), 6.84 (s, 1 H), 4.72(t, J=8.3 Hz, 1 H, 17α-H), 4.13 (q, J=7.0 Hz, 2 H), 2.64-2.52 (m, 3 H),2.29 (s, 3 H, COCH₃), 2.07 (s, 3 H, COCH₃), 2.23-1.26 (m, 18 H), 0.94(s, 3 H, CH₃); CIMS (isobutane) m/z (rel intensity) 429 (MH+, 49), 341(100). Anal. Calcd for C₂₅H₃₂O₆: C, 70.07; H, 7.53. Found: C, 70.42; H,7.75.

B-Homo-2-ethoxy-6-oxoestra-1,3,5(10)-trien-3,17β-diol (14). Undernitrogen, a suspension of compound 13 (0.95 g, 2.2 mmol) in anhydrousmethanol (10 ml) was cooled to −5-0° C. and then a 20% solution ofpotassium hydroxide in methanol (10 ml, KOH 1 g, 17.8 mmol) was addeddropwise. The resulting mixture was allowed to warn to room temperatureand stirred for 3.5 hr. The mixture was cooled to 0° C. and thenneutralized with 3 N HCl to pH 5, and allowed to precipitate in arefrigerator overnight. The resultant mixture was filtered to afford 14as a white solid (725 mg, 95%), which was recrystallized from methanolto afford an analytical sample: mp 198-200° C.; IR (KBr, cm⁻¹) 3497(OH), 3281 (OH), 2935, 2882, 1650 (6-C═O), 1608, 1508; ¹H NMR (CDCl₃)δ7.22 (s, 1 H, 4-ArH), 6.76 (s, 1 H, 1-ArH), 4.19 (q, J=7 Hz, 2 H,2-CH₂—), 3.78 (t, J=8.5 Hz, 1 H, 17α-H), 2.54 (m, 3 H), 2.15-1.21 (m, 18H), 0.90 (s, 3 H, 18-CH₃); CIMS (isobutane) m/z (rel intensity) 345(MH+, 100). Anal. Calcd for C₂₁H₂₈O₄: C, 73.23; H, 8.19. Found: C,73.40; H, 8.43.

B-Homo-3,17β-dihydroxy-2-ethoxyestra-1,3,5(10)trien-7-one (9);B-Homo-3,17β-dihydroxy-2-ethoxyestra-1,3,5(10)trien-6-one (14). Asolution of trimethylsilyldiazomethane (4.83 ml, 2 M in hexane, 9.7mmol) was added dropwise to a solution of ketone 5 (1 g, 2.41 mmol) indichloromethane (40 ml) containing BF₃.Et₂O (1.5 ml, 12.2 mmol) at −20°C. The reaction mixture was maintained at the same temperature for 3 hrand poured over ice/water (50 ml). The organic layer was dried (Na₂SO₄)and the solvent was removed. The residue was then dissolved in diethylether (40 ml), silica gel (20 g) and 6 M HCl (1 ml) were added and thereaction mixture was stirred at room temperature for 4 hr. Usual workupof the reaction mixture gave a sticky residue which was dissolved inanhydrous methanol (30 ml). A 20% solution of KOH in methanol (15 ml)was slowly added and the reaction mixture was stirred at roomtemperature for additional 4 hr. It was then neutralized with 6 M HCland the solvent was removed. Extraction of the crude product withorganic solvent followed by column chromatography(silica gel: 230-420mesh, 1:2 ethyl acetate-hexane) gave compound 14 (0.2 g, 24%) andcompound 9 (0.26 g, 31.3%). The IR and NMR spectra of these compoundswere identical with those of the samples described above.

B-Homo-2-ethoxy-3,17β-dihydroxyestra-1,3,5(10)-triene-7-one hydrazone(15). Under nitrogen and at room temperature, compound 9 (60 mg, 0.17mmol) was dissolved in anhydrous pyridine (5 ml) and then hydroxylaminehydrochloride (240 mg, 3.48 mmol) was added. The reaction mixture wasstirred at room temperature for 40 hr. The pyridine was removed underreduced pressure at 30-35° C. The residue was dissolved in ethyl acetate(50 ml) and water (30 ml) and the ethyl acetate was washed with brine(2×20 ml), dried over sodium sulfate, and evaporated to dryness.Chromatography of the residue (silica gel 230-400 mesh, ethylacetate:hexane 1.25:1 by volume) gave the compound 16 as off-whitecrystals (50.3 mg, 80.3%): mp 236-238° C.; IR (KBr, cm⁻¹) 3292 (br. OH,N═OH), 2930, 1737 (w, C═N), 1592, 1510; ¹H NMR (CDCl₃+DMSO-d₆) δ9.88 (s,1 H, C═NOH), 7.03 (s, 1 H, OH), 6.77 (s, 1 H,), 6.71 (s, 1 H), 4.12 (q,J=7.0 Hz, 2 H), 3.86 (d, J=20.1 Hz, 1 H), 3.68 (t, J=8.5 Hz, 1 H), 3.43(d, J=19.8, 1 H), 2.20-1.10 (m, 16 H), 0.84 (s, 3 H); CIMS (isobutane)m/z (rel intensity) 514 (MH+, 1.10). Anal. Calcd for C₂₈H₃₆O₅N₂S: C,65.6; H, 7.08; N, 5.46. Found: C, 65.80; H, 7.33; N, 5.35.

B-Homo-2-ethoxyestra-1,3,5(10),6-tetraene-3,17,β-diol (16). A solutionof methyllithium in THF/cumene (1.0 M, 2 ml, 2.0 mmol) was addeddropwise to a stirred solution of tosylhydrazone 10 (75 mg, 0.15 mmol)in THF (10 ml) under argon at 0° C. The reaction mixture was allowed towarm to room temperature after 2 hr. After stirring a further 20 hr atroom temperature, the reaction mixture was poured into ice (50 g) andacidified with 6 N HCl. The resultant mixture was extracted with ethylacetate (3×30 ml) and the organic extracts were washed with saturatedsodium sulfate (30 ml) and brine (2×30 ml), combined and dried oversodium sulfate, and evaporated to dryness. Chromatography of the residueon silica gel (230-400 mesh) using ethyl acetate:hexane 2:5 by volumegave the compound 16 (26 mg, 54%), which was crystallized from ethylacetate/hexane to afford white crystals: mp 167-168° C.; IR (KBr, cm⁻¹)3503 (OH), 3231 (OH), 2930, 1603 (w, C═C), 1510; ¹H NMR (CDCl₃) δ6.82(s, 1 H), 6.70 (s, 1 H), 6.54 (d, J=10.1 Hz, 1 H), 6.06 (m, 1 H), 5.51(s, OH, 1 H), 4.15 (q, J=7.0 Hz, 2 H), 3.74 (t, J=8.7 Hz, 1 H, 17α-H),2.12-1.21 (m, 14 H), 0.84 (s, 3 H, CH₃); CIMS (isobutane) m/z (relintensity) 329 (MH+, 100), 311 (52). Anal. Calcd for C₂₁H₂₈O₃.1/4H2O: C,75.58; H, 8.84. Found: C, 75.62; H, 8.65.

B-Homo-17β-acetoxy-2-ethoxy-3-hydroxyestra-1,3,5(10)-trien-6-one (18).Under nitrogen, a solution of compound 13 (65 mg, 0.15 mmol) in methanol(10 ml) was deoxygenated by bubbling through it a slow stream ofnitrogen for 30 min. A similarly deoxygenated solution of KHCO₃ (152 mg,1.52 mmol) in water (1 ml) was added and the reaction mixture wasstirred and heated at 65° C. (outer bath) for 1.5 hr. The reactionmixture was cooled to room temperature and then neutralized to pH=6 with6 N HCl. The solvents were removed under reduced pressure and theresidue was dissolved in a mixture of ethyl acetate (50 ml) and water(50 ml). The ethyl acetate layer was separated, dried over sodiumsulfate and evaporated to dryness. Chromatography of the residue (silicagel 230-400 mesh, ethyl acetate:hexane 1:2 by volume) gave themonoacetate 18 (56 mg, 95%), which was recrystallized from ethylacetate/hexane to afford white crystals: mp 240-242° C.; IR (KBr, cm⁻¹)3385 (OH), 2941, 1721 (17-C═O), 1663 (6-C═O), 1614, 1511; ¹H NMR (CDCl₃)δ7.22 (s, 1 H, 4-ArH), 6.75 (s, 1 H, 1-ArH), 5.54 (s, 1 H, 3-ArOH), 4.72(t, J=8.5 Hz, 1 H, 17α-H), 4.19 (q, J=7 Hz, 2 H, 2-CH₂—), 2.58 (m, 3 H),2,55 (m, 1 H), 2.08 (s, 3 H, 3-CH₃CO), 1.94-1.27 (m, 11 H), 0.88 (s, 3H, 18-CH₃) CIMS (isobutane) m/z (rel intensity) 387 (MH+, 100); Anal.Calcd for C₂₃H₃₀O₅: C, 71.48; H, 7.82. Found C, 71.22; H, 7.86.

B-Homo-3-Acetoxy-2-ethoxy-17β-hydroxyestra-1,3,5(10)-trien-6-one(19).Under argon and at room temperature, diol 14 (60 mg, 0.17 mmol) wasdissolved in THF (4 ml) and 1 N NaOH (0.19 ml, 0.19 mmol) was added tothe solution. After stirring for 20 min, a solution of 1-acetyl-1H-1,2,3-triazolo[4,5-b]pyridine (31 mg, 0.19 mmol) in THF (2 ml) wasadded dropwise to the above reaction mixture. The reaction mixture wasstirred at room temperature for 1.5 hr. The reaction mixture was pouredinto ice (20 g) and neutralized to pH 6 with 2 N HCl, and then extractedwith ethyl acetate (2×30 ml). The ethyl acetate layers were combined,dried over sodium sulfate and evaporated to dryness. Chromatography ofthe residue (silica gel 230-400 mesh, ethyl acetate:hexane =1:3 byvolume) gave compound 19 (61 mg, 90%), which was obtained as a whitefoam by evaporation of a hexane solution under high vacuum at 40-50° C.;IR (KBr, cm⁻¹) 3449 (br. OH), 2933, 1766 (3-C═O), 1669 (6-C═O), 1605,1502; ¹H NMR (CDCl₃) δ7.36 (s, 1 H, 4-Ar—H), 6.86 (s, 1 H, 1-Ar—H), 4.14(q, J=7 Hz, 2 H, 2-CH₂—), 3.78 (t, J=8.5 Hz, 1 H, 17α-H), 2.59 (m, 3 H),2.55 (m, 1H), 2.30 (s, 3 H, 17-CH₃CO), 2.15-1.27 (m, 11 H), 0.89 (s, 3H, 18-CH₃); CIMS (isobutane) m/z (rel intensity) 387 (MH+, 100); Anal.Calcd for C₂₃H₃₀O₅: C, 71.48; H, 7.82. Found C, 71.60; H, 8.16.

B-Homo-2-ethoxy-6-methoximinoestra-1,3,5(10)-trien-3,17β-diol (20). To asolution of diacetate 13 (76 mg, 0.21 mmol) in pyridine (10 ml) wasadded methoxylamine hydrochloride (354 mg, 4.23 mmol) in one portionunder nitrogen. The resulting mixture was heated at 100° C. for 3 hr andthen cooled to about 50° C. The pyridine was removed under reducedpressure. The residue was dissolved in a mixture of ethyl acetate (50ml) and water. The ethyl acetate solution was separated and then washedwith brine (2×30 ml), dried over sodium sulfate, and evaporated todryness. Chromatography of the residue (silica gel 230-400 mesh, ethylacetate:hexane 3:5 by volume) gave compound 20 (78 mg, 94%), which wascrystallized from ethyl acetate/hexane to afford white crystals: mp181-183° C.; IR (KBr, cm ⁻¹) 3511 (OH), 3318 (OH), 2972, 2987,1618,1576, 1508; ¹H NMR (CDCl₃) δ7.03 (s, 1 H, 4-ArH), 6.74 (s, 1 H, 1-ArH),5.53 (s, 1 H, 3-ArOH), 4.17 (q, J=7 Hz, 2 H, 2-CH₂—), 4.14 (s, 3 H,-OCH₃), 3.78 (t, J=8.5 Hz, 1 H, 17α-H), 2.58 (m, 2 H), 1.94 (m, 3 H),1.78-1.27 (m, 15 H), 0.88 (s, 3 H, 18-CH3); CIMS (isobutane) m/z (relintensity) 374 (MH+, 100). Anal. Calcd for C₂₂H₃₁O₄N: C, 70.75; H, 8.37;N 3.75. Found: C, 70.75; H, 8.41; N 3.61.

B-Homo-3,17β-diacetoxy-2-ethoxy-6-methoximinoestra-1.3.5(10)-triene(21). Acetic anhydride (0.6 ml, 6.3 mmol) was added under argon at roomtemperature to a solution of compound 20 (46 mg, 0.12 mmol) in anhydrouspyridine (3 ml). The resulting mixture was stirred at room temperaturefor 20 hr and then poured into ice/water mixture (50 g). The compoundwas extracted with ethyl acetate (3×40 ml). The organic layers werewashed with sodium bicarbonate (50 ml) and brine (50 ml), dried oversodium sulfate and evaporated to dryness. Chromatography of the residueon a silica gel (230-400 mesh) column using ethyl acetate:hexane 1:5 byvolume gave the compound 27 (51 mg, 90%), which was dried under highvacuum at 40-50° C. to give the title compound 21 as a stable whitefoam: IR (KBr, cm⁻¹) 2934, 1770 (3-C═O), 1734 (17-C═O), 1614, 1500; ¹HMR (CDCl₃) δ7.12 (s, 1 H, 4-ArH), 6.81 (s, 1 H, 1-ArH), 4.72 (t, J=8.5Hz, 1 H, 17α-H), 4.08 (q, J=7 Hz, 2 H, 2-CH₂—), 3.96 (s, 3 H, —OCH₃),2.57 (m, 2 H), 2.38 (s, 3 H, 3-CH₃CO), 2.06 (s, 3 H, 17-CH₃CO),2.32-1.26 (m, 14 H), 0.90 (s, 3 H, 18-CH₃); CIMS (isobutane) m/z (relintensity) 458 (MH+, 100). Anal. Calcd for C₂₆H₃₅O₆N: C, 68.25; H, 7.71;N 3.06. Found C, 68.45; H, 7.87; N 2.79.

B-Homo-2-ethoxy-6-hydroximinoestra-1,3,5(10)-trien-3,17β-diol (22).Under nitrogen and at room temperature, compound 14 (200 mg, 0.58 mmol)was dissolved in anhydrous pyridine (10 ml) and then hydroxylaminehydrochloride (807 mg, 11.6 mmol) was added. The reaction mixture wasstirred at room temperature for 20 hr. The pyridine was removed underreduced pressure at 30-35° C. and the residue was dissolved in a mixtureof ethyl acetate (50 ml) and water (30 ml). The ethyl acetate layer wasseparated and washed with brine (2×20 ml), dried over sodium sulfate,and evaporated to dryness. Chromatography of the residue ( silica gel230-400 mesh, ethyl acetate : hexane 5:4 by volume) gave the titlecompound 22 (198 mg, 95%), which was recrystallized from ethyl acetateto afford white crystals: mp 226-228° C.; mg, 80.3%); IR (KBr, cm⁻¹)3452 (OH), 3160 (OH), 3039, 2932, 2897, 1722 (w, 6-C═N), 1599, 1150; ¹HNMR (CDCl₃+DMSO-d₆) δ9.73 (s, 1 H, C═NOH), 7.34 (s, 1 H), 6.73 (s, 1 H),6.17 (s, 1 H, OH), 4.15 (q, J=7.0 Hz, 2 H), 3.86 (d, J=20.1 Hz, 1 H),3.73 (t, J=8.5 Hz, 1 H), 2.63-1.17 (m, 19 H), 0.86 (s, 3 H); CIMS(isobutane) m/z (rel intensity) 360 (MH+, 100). Anal. Calcd forC₂₁H₂₉O₄N: C, 70.17; H, 8.13; N, 3.90. Found: C, 70.16; H, 8.45; N,3.69.

B-Homo-6β-actamido-3,17α-diacetoxy-2-ethoxyestra-1,3,5(10)-triene (23).

To a solution of compound 14 (270 mg, 0.78 mmol) and ammonium acetate(4.0 g, 51.9 mmol ) in anhydrous methanol (20 ml) was added sodiumcyanoborohydride (493 mg, 7.8 mmol) at room temperature under argon. Thereaction mixture was stirred at room temperature for 30 min and thenrefluxed at 65-70° C. for 48 hr. The mixture was cooled to about 40° C.and then the solvent was removed under reduced pressure. The residue was treated with saturated NaHCO₃ (50 ml) and ethyl acetate (3×50 ml).The ethyl acetate layers were washed with brine (2×30 ml), combined anddried over sodium sulfate, and evaporated to dryness. The residue wasdissolved in anhydrous pyridine (10 ml) and acetic anhydride (4 ml) wasadded, and the resultant mixture was stirred at room temperature underargon for 14 hr. The pyridine and excess acetic anhydride were removedunder reduced pressure at 40-45 ° C. and the residue dissolved in ethylacetate (100 ml). The ethyl acetate solution was washed with saturatedNaHCO₃ (50 ml) and brine (50 ml), dried over sodium sulfate, andevaporated to dryness. Chromatography of the residue on silica gel(230-400 mesh) using ethyl acetate:hexane 7:2 by volume gave thecompound 23 (99 mg, 27%), which was dried under high vacuum at 40-50° C.to give stable white foam: IR (KBr, cm⁻¹) 3313 (6-NH), 2935; 1765(3-C═O) 1733 (17-C═O), 1655 (6-NC═O), 1510; ¹H NMR (CDCl₃) δ6.95 (s, 1H, 4-ArH), 6.85 (s, 1 H, 1 H-ArH), 5.80 (d, J_(NH-6)=7.2 Hz, 6β-CNH, 1H), 5.13 (ddd, J_(NH-6)=7.2 Hz, J₆₋₇ =11.7 and 6.4 Hz, 6α-CH), 4.73 (t,J=8.5 Hz, 1 H, 17α-H), 4.08 (qd, J=7.1 Hz, J=1.5 Hz, 2 H, 2-CH₂—), 2.56(m, 1 H), 2.28 (s, 3 H, 3-CH₃CO), 2.07 (s, 3 H, 17-CH₃CO), 1.95 (s,6-NCOCH₃, 3 H), 2.36-1.26 (m, 14 H), 0.90 (s, 3 H, 18-CH₃); CIMS(isobutane) m/z (rel intensity) 472 (MH+, 100). Anal. Calcd forC₂₇H₃₇O₆N: C, 68.77; H, 7.91; N, 2.97. Found: C, 68.87; H, 8.15; N,2.87.

B-Homo-6β-actamido-3,17β-diacetoxy-2-ethoxyestra-1,3,5(10)-triene (24).The chromatographic purification of 23 described above also providedcompound 24 as a white foam (180 mg, 48%): IR (KBr, cm⁻¹) 3313 (6-NH),2935; 1765 (3-C═O), 1734 (17-C═O), 1655 (NC═O), 1510; ¹H NMR (CDCl₃)δ6.88 (s, 1 H, 4-ArH), 6.78 (s, 1 H 1-ArH), 5.68 (d, J_(NH-6)=8.5 Hz,6α-CNH, 1 H), 5.40 (ddd, J_(NH-6)=8.5 Hz, J₆₋₇=11.7 and 6.4 Hz, 6β-CH),4.69 (t, J=8.5 Hz, 1 H, 17α-H), 4.08 (qd, J=7 Hz, J=1.5 Hz, 2 H,2-CH₂—), 3.96 (s, 3 H, —OCH₃), 2.52 (m, 1 H), 2.30 (s, 3 H, 3-CH₃CO),2.10 (s, 3 H, 17-CH₃CO), 2.07 (s, 6-NCOCH₃, 3 H), 2.36-1.26 (m, 14 H),0.88 (s, 3 H, 18-CH₃); CIMS (isobutane) m/z (rel intensity) 472 (MH+,100). Anal. Calcd for C₂₇H₃₇O₆N: C, 68.77; H, 7.91; N, 2.97. Found: C,68.85; H, 8.15; N, 2.90.

B-Homo-6α-acetamido-2-ethoxyestra-1,3,5(10)-trien-3,17β-diol (25). Asolution of compound 23 (75 mg, 0.16 mmol) in methanol (14 ml) wasdeoxygenated by bubbling through it a slow stream of nitrogen for 30min. A similarly deoxygenated 1 M solution of sodium hydroxide in water(1.6 ml, 16 mmol) was added and the mixture stirred at room temperaturefor 5 hr. The reaction mixture was neutralized with acetic acid to pH 6,and the solvents were removed under reduced pressure. The residue wasdissolved in a mixture of ethyl acetate (60 ml) and saturated sodiumbicarbonate (50 ml). The organic layer was separated and then washedwith brine (2×30 ml), dried over sodium sulfate, and evaporated todryness. Chromatography of the residue on a silica gel column usingmethylene chloride:acetone 2:1 by volume gave the title compound 25 (42mg, 68%), which solidified from an acetone-hexane-methylene chloridemixture to afford a white solid: mp 196-198° C.; IR (KBr, cm⁻¹) 3328(OH), 3313 (6-NH), 2937; 1650 (6-NC═O), 1512; 1 H NMR (CDCl₃) δ6.84 (s,1 H, 4-ArH), 6.77 (s, 1 H, 1-ArH), 5.73 (d, J_(NH)-6=7.2 Hz, 6β-CNH, 1H), 5.57 (s, OH, 1H), 5.08 (ddd, J_(NH-6)=7.2 Hz, J₆₋₇=11.7 and 6.4 Hz,6α-CH), 4.08 (qd, J=7 Hz, J=1.5 Hz, 2 H, 2-CH2—), 3.75 (t, J=8.5 Hz, 1H, 17α-H), 2.50 (m, 1 H), 2.33 (m, 1 H), 1.95 (s, 6-NCOCH3, 3 H),2.18-1.26 (m, 17 H), 0.85 (s, 3 H, 18-CH3); CIMS (isobutane) m/z (relintensity) 388 (MH+, 100). Anal. Calcd for (C₂₃H₃₃O₄N.2/3CH₃COCH₃): C,70.44; H, 8.75; N 3.28. Found: C, 70.60; H, 9.14; N, 3.59.

B-Homo-6β-acetamido-2-ethoxyestra-1,3,5(10)-trien-3,17β-diol (26).Compound 24 (178 mg, 0.38 mmol) was treated by the method describedabove for the preparation of 25 to give 26 (98 mg, 67%), whichsolidified from acetone-hexane-methylene chloride to afford a whitesolid: mp>162° C. (dec.); IR (KBr, cm⁻¹) 3314 (OH, 6-NH), 2927; 1658(6-NC═O), 1506; ¹H NMR (CDCl₃) δ6.78 (s, 1 H, 4-ArH), 6.72 (s, 1 H,1-ArH), 5.71 (d, J_(NH)-6=8.8 Hz, 6α-CNH, 1 H), 5.61 (s, OH, 1 H), 5.38(ddd, J_(NH)-6=8.8 Hz, J₆₋₇=11.7 and 6.4 Hz, 6β-CH), 4.11 (qd, J=7.1 Hz,J=1.5 Hz, 2 H, 2-CH₂—), 3.75 (t, J=8.5 hz, 1 H, 17α-H), 2.48 (m, 1 H),2.10 (s, 6-NCOCH₃, 3 H), 2.18-1.26 (m, 18 H), 0.83 (s, 3 H, 18-CH₃);CIMS (isobutane) m/z (rel intensity) 388 (MH+, 100). Anal. Calcd forC₂₃H₃₃O₄N: C, 71.29; H, 8.58; N 3.61. Found: C, 71.09; H, 8.85; N, 3.49.

17β-Acetoxy-2-ethoxy-6-hydroximinoestra-1,3,5(10)-triene-3-ol (27). Asolution of 3,17,β-diacetoxy-2-ethoxy-estra-1,3,5(10)-triene-6-one (5,800 mg, 1.93 mmol) in pyridine (12 ml) was treated with hydroxylaminehydrochloride (1.07 g, 15.4 mmol). The resulting mixture was stirred andheated at 100° C. for 30 min. The mixture was cooled to room temperatureand then poured into ice/water mixture (150 ml). The compound wasextracted with ethyl acetate (3×100 ml). The combined organic layerswere washed with sodium bicarbonate (100 ml), water (2×100 ml) and brine(2×100 ml), dried over sodium sulfate, and evaporated to dryness.Chromatography of the residue on silica gel using 30% ethyl acetate inhexane gave the title compound (763 mg, 89%), which was crystallizedfrom ethyl acetate/hexane to afford title compound 27 as yellowishcrystals: mp 226-227° C. ¹H NMR (CDCl₃) δ7.55 (s, 1 H). 6.78 (s, 1 H),4.75 (t, J=8.5 Hz, 1 H), 4.19 (q, J=6.9 Hz, 2 H), 3.15 (dd, J=4.3 and 12Hz, 1 H), 1.2-2.40 (m, 18 H), 0.85 (s, 3 H); CIMS (isobutane) m/z (relintensity) 388 (MH+, 100), 372 (MH—H₂O, 10), 328 (75); Anal. Calcd forC₂₂H₂₉O₅N: C, 68.20; H, 7.54; N, 3.61. Found: C, 68.04; H, 7.62; N,3.54.

B-Homo-6-aza-2-ethoxy-3,17β-dihydroxyestra-1,3,5(10)-trien-7-one (29).To a solution of 27 (700 mg, 1.63 mmol) in pyridine (10 ml) was addedp-toluenesulfonyl chloride (686 mg, 3.6 mmol). The resulting mixture wasstirred at room temperature for 2 hr, and the pyridine was then removedin vacuo at ambient temperature. The residue, containingO-toluenesulfonyloxime 28, was dissolved in 40% chloroform in benzene (5ml) and the material was applied to the top of a basic alumina column(30×3.5 cm). The column was eluted with chloroform/benzene (40-90%chloroform) and then allowed to stand overnight. The column was theneluted with methanol (200 ml) and then with 80% methanol in water (200ml). The methanol eluant was collected and evaporated to dryness.Chromatography of the residue on a silica gel column using ethyl acetategave the title compound 29 (267 mg, 47%), which was crystallized fromethyl acetate to afford white crystals: mp 222-223° C. ¹H NMR (CDCl₂)δ7.31 (br.s., N—H, 1 H). 7.55 (s, 1 H, 4-aromatic CH), 6.78 (s, 1 H,1-aromatic CH), 4.19 (q, J=6.9 Hz, 2 H, 2-CH₂—), 3.75 (t, J=8.5 Hz, 1 H,17a-H), 2.41 (m, 2 H, 7-CH₂), 1.43 (t, J=7 Hz, 3 H, 2-CH₃), 0.82 (s, 3H, 18-CH₃); CIMS (isobutane) m/z (rel intensity) 346 (MH⁺, 100), 328(MH—H₂O, 20); Anal. Calcd for C₂₀H₂₇O₄N.1/3 H₂O: C, 68.35; H, 7.93; N,3.98. Found: C, 68.25; H, 8.03 N, 3.82.

B-Homo-6-aza-2-ethoxyestra-1,3,5(10)-triene-3,17β-diol (30). A solutionof borane in THF (1.0 M, 5.6 ml, 5.6 mmol) was added dropwise by syringeto a solution of the lactam 29 (97.8 mg, 0.28 mmol) in THF (5 ml) underargon at room temperature, and the resulting solution was stirred for 6hr and then at gentle reflux for 4 hr. The mixture was cooled and 6 Nhydrochloric acid (2 ml) was added slowly through a pipette. The solventwas removed under reduced pressure and the residue was dissolved inethyl acetate (60 ml). The ethyl acetate solution was washed with sodiumbicarbonate (30 ml) and brine (2×20 ml), dried over sodium sulfate andevaporated to dryness. Chromatography of the residue on silica gel using50% ethyl acetate in methylene chloride gave the title compound 30 (85mg, 90%), which formed a stable white foam when a methylene chloridesolution was evaporated under reduced pressure. ¹H NMR (CDCl₃) δ6.80 (s,1 H), 6.28 (s, 1 H), 4.05 (q, J=6.9 Hz, 2 H), 3.75 (t, J=8.5 Hz, 1 H),3.30 (t, J=6.9 Hz, 1 H), 2.82 (t, J=6.9 Hz, 1 H), 2.55 (dt, J=8.5 Hz,J=2 Hz, 1 HH, 9a-H), 1.2 -2.20 (m, 18 H), 0.78 (s, 1 H); CIMS(isobutane) m/z (rel intensity) 332 (MH⁺, 100), 314 (MH—H₂O, 60); Anal.Calcd for C₂₀H₂₉O₃N.1/4 H₂O: C, 71.50; H, 8.85; N, 4.14. Found: C,71.64; H, 8.90; N, 4.29.

B-Homo-6-acetyl-6-aza-3,17β-diacetoxy-2-ethoxyestra-1,3,5(10)-triene(31). Under nitrogen, acetic anhydride (1.83 g, 1.7 ml, 17.65 mmol) wasadded to a solution ofB-homo-6-aza-2-ethoxyestra-1,3,5(10)-triene-3,17β-diol (30, 146 mg, 0.44mmol) in pyridine (5 ml). The resulting mixture was stirred at roomtemperature for 24 hr and then poured into ice/water mixture (30 ml).The compounds were extracted with ethyl acetate (3×50 ml) and theorganic layers were washed with water (30 ml), saturated sodiumbicarbonate (30 ml) and brine (30 ml), dried over sodium sulfate, andevaporated to dryness. Chromatography of the residue on silica gel using33% ethyl acetate in methylene chloride gave the title compound 31 (169mg, 84%), which formed a white stable foam when an ethyl acetate/hexanesolution was evaporated under reduced pressure. ¹H NMR (CDCl₃) δ6.87 (s,1 H), 6.78 (s, 1 H), 4.66 (t, J=8.5 Hz, 1 H, 17a-H), 4.09 (q, J=6.9 Hz,2 H, 2-CH₂—), 4.08 (t, J=6.9 Hz, 1 H), 3.09 (m, 1 H), 2.46 (dt, J=8.5Hz, J=2 Hz, 1 H, 9a-H), 2.4 (s, 3 H, 3-CH₃CO), 2.28 (s, 3 H, 17-CH₃CO),1.93 (s, 3 H, N—CH₃CO), 0.78 (s, 3 H=6.9 Hz, 1 H); CIMS (isobutane) m/z(rel intensity) 458 (MH⁺, 100), 398 (35); Anal. Calcd for C₂₆H₃₅O₆N: C,68.25; H, 7.71; N, 3.06. Found: C, 68.36; H, 7.90; N, 3.43.

B-Homo-6-acetyl-6-aza-2-ethoxyestra-1,3,5(10)-triene-3,17β-diol (32). Asolution of 31 (150 mg, 0.33 mmol) in methanol (15 ml) was deoxygenatedby bubbling through it a slow stream of nitrogen for 30 min. A similarlydeoxygenated solution of 1.0 N sodium hydroxide in water (3.2 ml, 3.2mmol) was added and the mixture stirred at room temperature for 4.5 hr.The reaction mixture was neutralized with acetic acid to pH 6-7 and thenthe solvents were removed under reduced pressure. The residue wasdissolved in ethyl acetate (60 ml) and the organic layer was washed withwater (30 ml), sat. sodium bicarbonate (30 ml) and brine (30 ml), driedover sodium sulfate, evaporated to dryness. Chromatography of theresidue on silica gel using ethyl acetate gave the title compound 32(105 mg, 86%), which was crystallized from ethyl acetate/hexane toafford a white crystals: mp 189-191° C.; ¹H NMR (CDCl₃) δ6.77 (s, 1 H),6.67 (s, 1 H), 4.09 (q, J=6.9 Hz, 2 H, 2-CH₂—), 4.08 (t, J=6.9 Hz, 1 H),3.71 (t, J=8.5 Hz, 1 H, 17a-H), 3.03 (m, 1 H), 2.40 (dt, J=8.5 Hz, J=2Hz, 1 H, 9a-H), 1.87 (s, 3 H. CH₃CON), 0.78 (s, 1 H); CIMS (isobutane)m/z (rel intensity) 374 (MH⁺, 100); Anal. Calcd for C₂₂H₃₁O₄N.1/3 H₂O:C, 69.75; H, 8.41; N, 3.69. Found: C, 69.65; H, 8.25; N, 3.87.

B-Homo-6-aza-2-ethoxy-6-ethylestra-1,3,5(10)-triene-3,17β-diol (33). Toa solution ofB-homo-6-acetyl-6-aza-2-ethoxyestra-1,3,5(10)-triene-3,17β-diol (32, 90mg, 0.24 mmol) in THF (5 ml) was added dropwise a solution of borane inTHF (1.0 M, 4.8 ml, 4.8 mmol) by syringe under argon. The resultingsolution was stirred at room temperature for 4 hr and then at gentlereflux for 4 hr. The mixture was cooled to room temperature and 6 Nhydrochloric acid (1 ml) was added slowly through a pipette. The solventwas removed under reduced pressure and the residue was neutralized withsaturated sodium bicarbonate solution and then extracted with ethylacetate (3×30 ml). The ethyl acetate layers were washed with brine (2×30ml), dried over sodium sulfate and evaporated to dryness. Chromatographyof the residue on silica gel using 20% ethyl acetate in methylenechloride gave the title compound 33 (70 mg, 81%), which formed a stablewhite foam when an ethyl acetate/hexane solution was evaporated underreduced pressure: ¹H NMR (CDCl₃) δ6.68 (s, 1 H), 6.51 (s, 1 H), 4.07 (q,J=6.9 Hz, 2 H), 3.78 (t, J=8.5 Hz, 1 H), 3.38 (td, J=11.5 Hz, J=2.2 Hz,1 H), 3.2 (qd, J=12.6 Hz, J=7.1 Hz, 1 H) ,2.94 (qd, J=11.7 Hz, J=6.8 Hz,1 H), 2.75 (td, J=11.5 Hz, J=2.2 Hz, 1 H), 2.55 (dt, J=8.5 Hz, J=2 Hz, 1H, 9a-H), 1.2-2.20 (m, 18 H), 0.84 (s, 3 H); CIMS (isobutane) m/z (relintensity) 360 (MH⁺, 100), 342 (MH—H₂O, 50); Anal. Calcd forC₂₂H₃₃O₃N.1/6 H₂O: C, 72.96; H, 9.27; N, 3.86. Found: C, 72.97; H, 9.27;N, 3.85.

B-Homo-2-ethoxyestra-3,17β-triol (34). Under argon, sodium borohydride(260 mg, 7 mmol) is added to a solution of 9 (1.73 g, 5.02 mmol) inethanol. The mixture is stirred overnight at room temperature, then 6 Nhydrochloric acid is added dropwise with stirring until the pH is ca. 6.The solvent is removed under reduced pressure and the residue isdissolved in ethyl acetate (1.00 ml). The ethyl acetate solution iswashed with sodium bicarbonate (50 ml) and brine (2×25 ml), dried oversodium sulfate, and evaporated to dryness. Chromatography of the residueon silica gel using a gradient of ethyl acetate in methylene chloridegives the title compound 34 as a mixture of C-7 diastereomers.

B-Homo-3,17β-triacetoxy-2-ethoxyestra-1,3,5). Acetic anhydride (6 ml, 63mmol) is added under argon at room temperature to a solution of triol 34(1.10 g, 3.18 mmol) in anhydrous pyridine (12 ml). The resulting mixtureis stirred at room temperature for 24 hr and then poured into ice/watermixture (100 g). The compound is extracted with ethyl acetate (3×70 ml).The organic layers are washed with water (100 ml), aqueous sodiumbicarbonate (2×100 ml) and brine (2×100 ml), dried over sodium sulfate,and evaporated to dryness. Chromatography of the residue on silica gel(230-400 mesh) using ethyl acetate:hexane 1:1 by volume provides thetitle compound 35 as a mixture of C-7 diastereomers.

B-Homo-2-ethoxyestra-3,6,17β-triol (36). Under argon, sodium borohydride(260 mg, 7 mmol) is added to a solution of 14 (1.73 g, 5.02 mmol) inethanol. The mixture is stirred overnight at room temperature, then 6 Nhydrochloric acid is added dropwise with stirring until the pH is ca. 6.The solvent is removed under reduced pressure and the residue isdissolved in ethyl acetate (100 ml). The ethyl acetate solution iswashed with sodium bicarbonate (50 ml) and brine (2×25 ml), dried oversodium sulfate, and evaporated to dryness. Chromatography of the residueon silica gel using a gradient of ethyl acetate in methylene chloridegives the title compound 36 as a mixture of C-6 diastereomers.

B-Homo-3,17β-triacetoxy-2-ethoxyestra-1,3,5(10)-triene (37). Aceticanhydride (6 ml, 63 mmol) is added under argon at room temperature to asolution of triol 36 (1.10 g, 3.18 mmol) in anhydrous pyridine (12 ml).The resulting mixture is stirred at room temperature for 24 hr and thenpoured into ice/water mixture (100 g). The compound is extracted withethyl acetate (3×70 ml). The organic layers are washed with water (100ml), aqueous sodium bicarbonate (2×100 ml) and brine (2×100 ml), driedover sodium sulfate, and evaporated to dryness. Chromatography of theresidue on silica gel (230-400 mesh) using ethyl acetate:hexane 1:1 byvolume provides the title compound 35 as a mixture of C-6 diastereomers.

Anti-mitotic Activity In Vivo

Anti-angiogenic activity is evaluated in vivo by testing the ability ofa compound to inhibit the proliferation of new blood vessel cells(angiogenesis). A suitable assay is the chick embryo chorioallantoicmembrane (CAM) assay described by Crum et al., Science 230:1375 (1985).See also, U.S. Pat. No. 5,001,116, which is hereby incorporated byreference in its entirety. Briefly, fertilized chick embryos are removedfrom their shell on day 3 or 4, and a methylcellulose disc containingthe drug is implanted on the chorioallantoic membrane. The embryos areexamined 48 hours later and, if a clear avascular zone appears aroundthe disc, the diameter of that zone is measured. Using this assay, a 100mg disk of 2-methoxyestradiol is found to inhibit cell mitosis and thegrowth of new blood vessels after 48 hours.

Alternatively, anti-angiogenic activity can be assayed in vivo by the“mouse corneal micropocket assay” described by Kenyon et al., Opthalmol.& Visual Sci., 76:1625-1632 (1996); see also Klauber et al., Cancer Res57:81-86 (1997).

Tubulin polymerization Inhibition In Vitro

Potential anti-mitotic activity can be screened for by testing theability of a compound to inhibit tubulin polymerization and microtubuleassembly in vitro. Microtubule assembly is followed in a temperaturecontrolled recording spectrophotometer. For details and a discussion,see R. D'Amato et al., Proc Natl Acad Sci USA, 91:3964-3968 (1994).

A typical reaction mixture, which will have a final reaction volume of0.25 μl, contains 1.0 M monosodium glutamate (pH 6.6), 1.0 mg/ml (10 μM)tubulin, 1.0 MM MgCl₂, 4% (v/v) dimethylsulfoxide, and a compound to betested at the desired concentration (typically 0.1 to 100 μM). Thereaction mixtures are incubated for 15 min. at 37° C. and then chilledon ice. After addition of 10 μl 12.5 mM GTP (and, optionally, 0.33 mg/mlmicrotubule-associated proteins, or MAPs) to initiate polymerization,each reaction mixture is transferred to a cuvette at 0° C., and abaseline established. At time zero, the temperature controller of thespectrophotometer is set to a target temperature, typically between 25and 37° C. Microtubule assembly is monitored by observing an increase inturbidity at 350 nm as the temperature of the reaction mixture rises tothe set target temperature.

The presence or absence of MAPs appears to affect the mechanism ofpolymerization to some extent, since the relative potencies of taxotereand certain compounds of this invention are dependent on the presence ofMAPs.

The results are presented in Table 1 as IC₅₀ values (μM) for inhibitionof tubulin polymerization. Alternatively, inhibition of microtubuleassembly can be detected by transmission electron microscopy, asdescribed in U.S. Pat. No. 5,504,074 which is hereby incorporated byreference in its entirety.

In those cases where acceleration of tubulin polymerization is observed,an IC₅₀ value for inhibition of polymerization is of course notderivable. Results for such compounds may be presented in the form ofturbidimetric curves, e.g. as presented in FIGS. 4 and 5 forrepresentative compounds 13 and 18. The diacetate 13 had paclitaxel-likeproperties in both glutamate- and MAP-induced assembly reactions,whereas the diol 14 was inactive. Removal of the C-17 O-acetyl groupyielded the inactive compound 19, but activity was largely retained uponselective removal of the C-3 O-acetyl group (compound 18). Thepaclitaxel-like properties of compounds 13 and 18 included more rapidinduction of polymer at reduced temperatures (25-26° C.), formation ofmicrotubules stable to disassembly at 0° C., reduction of the tubulincritical concentration, and polymer formation in 0.1 M4-morpholineethanesulfonate in the absence of MAPs. In contrast toobservations with paclitaxel (Grover et al., Biochemistry 34:3927-3934(1995)), tubulin assembly has not been observed in the absence of GTPwith either 13 or 18. Although inhibition of [³H]paclitaxel binding totubulin polymer by compound 13 is not substantial (maximal inhibitionobserved was about 20%), it is reproducible, suggesting that thecompound binds in or close to the paclitaxel site on tubulin polymers.

As shown in FIG. 4, in experiments with 0.75 mg/ml MAPs and 1.0 mg/mltubulin, compounds 13 and 18 enhanced tubulin assembly at 25° C.,although their activities appeared to be quantitatively lower than hadbeen the case in the glutamate reaction condition. A progressiveincrease in activity with up to 40 μM agent was seen with both compounds(data presented in full only for compound 13), and at all concentrationscompound 18 was less stimulatory than 13 (FIG. 4 presents data withcompound 18 only at 10 μM). In addition, FIG. 4 demonstrates the effectof 10 μM paclitaxel on the assembly reaction. With a temperature jumpdirectly from 0 to 25° C., the assembly reaction with paclitaxel wasmuch more extensive than that with compound 13. In part this could beattributed to assembly stimulated by paclitaxel that occurred prior totemperature equilibration, and this could be clearly demonstrated byadding a 10° C. step to the reaction sequence, as was done in theexperiment presented in FIG. 4.

It was possible that compounds 13 and 18 were not actually acting likepaclitaxel in enhancing microtubule assembly and in stabilizing themicrotubules formed in the course of the turbidity studies shown in FIG.4, but that these agents were causing formation of polymers of aberrantmorphology and temperature stability different from microtubules, suchas occurs with vinca alkaloids. Polymer morphology was examined byelectron microscopy. With compound 13 abundant microtubules were formedthat were indistinguishable from those formed in the absence of drug.Even more important, when the reaction mixtures were returned to 0° C.for 15 min, no microtubules were visualized on grids prepared from thereaction mixture without drug. In the presence of 40 μM 13 (FIG. 5) or10 μM paclitaxel abundant cold stable microtubules were observed.

When either MAPs or GTP was omitted from the reaction mixture, with 10μM tubulin no reaction occurred with either no drug or 40 μM compound13. In contrast, with 10 μM paclitaxel a reaction was observed in bothcases.

Quantitation of the Effects of Paclitaxel and Compound 13 on TubulinPolymerization.

FIG. 4: Each reaction mixture contained 0.8 M glutamate (pH 6.6), 0.4 mMGTP, 12 μM (1.2 mg/ml) tubulin, 4% (v/v) dimethyl sulfoxide, andcompound 13 or 18 as indicated. Baselines were established at 0°, and attime zero the temperature controller was set at 26°. At the timeindicated by the dashed line, the temperature controller was set at 0°.A. Curve 1, no drug; curve 2, 2 μM compound 13; curve 3, 5 μM 7; curve4, 10 μM 7; curve 5, 40 μM 7; curve 6, 40 μM 14. B. Curve 1, no drug;curve 2, 2 μM compound 18; curve 3, 5 μM 18; curve 4, 10 μM 18; curve 5,40 μM 18.

FIG. 5: Each reaction mixture contained 0.1 M Mes (pH 6.9 with NaOH in0.5 M stock solution), 0.1 mM GTP, 10 μM (1.0 mg/ml) tubulin, 0.75 mg/mlheat-treated MAPs, 4% dimethyl sulfoxide, and drug as indicated.Baselines were established at 0°, with drug added last, and the cuvettecontents were observed for 10 min. There was no change in turbidity inany reaction mixture. At this point the temperature controller was setat 10°, and subsequent temperature changes, as indicated, were made atthe times indicated by the vertical dashed lines. Curve 1, no drug;curve 2, 10 μM paclitaxel; curve 3, 10 μM compound 13; curve 4, 20 μM 7;curve 5, 40 μM 7; curve 6, 10 μM compound 18.

It is clear from FIG. 5 that the activities of 13 and 18 aresignificantly less than that of paclitaxel. It would be desirable toquantitate this difference, however 13 only minimally inhibits thebinding of [³H]paclitaxel to tubulin polymer, and 13 was inactive inroom temperature glutamate-dependent assay systems where nopolymerization reaction occurs in the absence of drug.

With 10 μM 13 and 40 μM tubulin, assembly of microtubules was observedwithout MAPs but not without GTP. Measurement of the criticalconcentration with and without MAPs with paclitaxel, and again with 13,was therefore carried out to provide some idea of the comparativeactivity of these agents. The data are presented in FIG. 6, which yieldcritical concentrations, obtained at 30° C., of 1.4, 0.06, and 0.50mg/ml for MAP-and GTP-dependent assembly without drug and in thepresence of 10 μM paclitaxel or 10 μM 13, respectively, and 0.20 and 1.4mg/ml for GTP-dependent, MAP-independent assembly with 10 μM paclitaxelor 10 μM 13. No reaction occurred without drug at tubulin concentrationsup to 4 mg/ml. (A constant weight ratio of MAPs to tubulin of 1:3 wasused, because the suboptimal concentration of MAPs caused greaterdifferences in the critical concentrations obtained.) Reaction mixtures(0.25 ml) contained 0.1 M Mes (pH 6.9), 100 μM GTP, 4% dimethylsulfoxide, the indicated tubulin concentration (with heat-treated MAPsin 1:3 weight ratio to the tubulin, panel A only), and either 10 μMpaclitaxel (▴), 10 μM compound 13 (◯), or no drug (▾). Cuvette contentswere equilibrated at 0°, drugs were added, temperature was jumped to 30°(about one min), and the turbidity changes after a 20 min incubationmeasured.

At first glance, the data suggest that the affinity of paclitaxel fortubulin polymers is 7-8-fold greater than that of compound 13, but thismethod may significantly underestimate the difference between the twocompounds. Previous observations with paclitaxel analogs indicate thatdifferences between compounds are magnified in restrictive reactionconditions and minimized in favorable reaction conditions. For example,it has been found that sarcodictyin A is significantly less potent thanpaclitaxel in its interactions with tubulin under restrictive reactionconditions, and it only weakly inhibits the binding of paclitaxel totubulin polymer (Hamel et al., Biochemistry 38:5490-5498 (1999)).However, under favorable reaction conditions (room temperature) thequantitative difference between paclitaxel and sarcodictyin A was only2.5-fold. Compound 13 was inactive in this room temperature assay. Thecritical concentration with 10 μM sarcodictyin A under the reactionconditions shown in FIG. 6B is 1.0 mg/ml in the GTP-only system.Relative order of compound activity thus persists, with sarcodictyin Abeing about 5-fold less active than paclitaxel and 40% more active than13.

Cytotoxicity Against Tumor cells in Vitro.

The compounds of the invention were tested for inhibition of cell growthin the established tumor cell screen of the National Cancer InstituteDevelopmental Therapeutics Program. See Boyd et al., Drug Dev. Res.,34:91-109 (1995). Results are presented in Table 1 as the “MGM” values(mean graph midpoint, in μM, for all cell lines tested).

TABLE 1 Tubulin polymerization Cytotoxicity inhibition Compound No. (MGMμM) (IC₅₀ μM) 9 n.t. >40 10 n.t. >40 11 n.t. 1.3 12 n.t. >40 13 n.t. *14 n.t. >40 18 n.t. * 19 n.t. >40 27 0.567 >40 28 n.t. n.t 29 16.2 4 3049.0 >40 31 38.0 >40 32 35.5 25 33 50.1 >40 n.t.: not tested *:Accelerates tubulin polymerization

While the examples presented above describe a number of embodiments ofthis invention, it is apparent to those skilled in the relevant artsthat the compounds, compositions, and methods of this invention can bealtered to provide alternative embodiments, and equivalent compositionsand methods, which nonetheless remain within the scope of thisinvention. Therefore, it will be appreciated that the present inventionis not limited in scope by the specific embodiments described above,which are intended only as illustrations of individual aspects of theinvention. In particular, modifications which are obvious to those ofordinary skill in the art are intended to be within the spirit and scopeof the following claims.

We claim:
 1. A compound of formula

wherein: A is selected from the group consisting of CH₂, NR⁴, C═O,C═NOH, CHNHCR⁵ and CHOH; B is selected from the group consisting of CH₂and C═O; R¹ is selected from the group consisting of H, methyl, ethyl,n-propyl, i-propyl, cyclopropyl, cyclopropylmethyl, 1-propenyl, allyl,and vinyl; R² and R⁴ are selected from the group consisting of H,methyl, ethyl, n-propyl, i-propyl, acetyl, propionyl, butyryl,cyclopropanecarbonyl, and isobutyryl; R³ is selected from the groupconsisting of H, acetyl, propionyl, butyryl, cyclopropanecarbonyl, andisobutyryl; and R5 is selected from the group consisting of H, methyl,ethyl, n-propyl, i-propyl, acetyl, propionyl, butyryl,cyclopropanecarbonyl, and isobutyryl.
 2. The compound of claim 1 whereinR¹ is selected from the group consisting of H, methyl, and ethyl.
 3. Thecompound of claim 2 wherein R²is selected from the group consisting ofH, acetyl, propionyl, butyryl, and isobutyryl.
 4. The compound of claim1 wherein R¹ is ethyl and R² is H or acetyl.
 5. The compound of claim 1wherein R³ is acetyl or propionyl.
 6. The compound of claim 2 wherein R³is acetyl or propionyl.
 7. The compound of claim 3 wherein R³ is acetylor propionyl.
 8. The compound of claim 4 wherein R³ is acetyl orpropionyl.
 9. The compound of any one of claims 5-8 wherein R3 isacetyl.
 10. The compound of any one of claims 1-8 wherein A is CH₂. 11.The compound of any one of claims 1-8 wherein A is C═O.
 12. The compoundof any one of claims 1-8 wherein A is NR⁴.
 13. The compound of claim 12wherein B is C═O.
 14. The compound of claim 12 wherein R⁴ is selectedfrom the group consisting of H, acetyl, and propionyl.
 15. The compoundof claim 14 wherein B is C═O.
 16. The compound of any one of claims 1-8wherein A is CHNHR⁴.
 17. The compound of claim 16 wherein R⁴ is selectedfrom the group consisting of H, acetyl, and propionyl.
 18. The compoundof claim 9 wherein A is CH₂.
 19. The compound of claim 9 wherein A isC═O.
 20. A method for treating a disease characterized by undesirablemitosis and/or undesirable angiogenesis, said method comprisingadministering to a mammal having said disease an effective amount of acompound of the formula below:

wherein: A is selected from the group consisting of CH₂, NR⁴, C═O,C═NOH, CHNHCR⁵ and CHOH; B is selected from the group consisting of CH₂and C═O; R¹ is selected from the group consisting of H, methyl, ethyl,n-propyl, i-propyl, cyclopropyl, cyclopropylmethyl, 1-propenyl, allyl,and vinyl; R² and R⁴ are selected from the group consisting of H,methyl, ethyl, n-propyl, i-propyl, acetyl, propionyl, butyryl,cyclopropanecarbonyl, and isobutyryl; R³ is selected from the groupconsisting of H, acetyl, propionyl, butyryl, cyclopropanecarbonyl, andisobutyryl; and R5 is selected from the group consisting of H, methyl,ethyl, n-propyl, i-propyl, acetyl, propionyl, butyryl,cyclopropanecarbonyl, and isobutyryl.
 21. The method of claim 20 whereinR¹ is selected from the group consisting of H, methyl, and ethyl. 22.The method of claim 21 wherein R² is selected from the group consistingof H, acetyl, propionyl, butyryl, and isobutyryl.
 23. The method ofclaim 20 wherein R¹ is ethyl and R² is H or acetyl.
 24. The method ofclaim 20 wherein R³ is acetyl or propionyl.
 25. The method of claim 21wherein R³ is acetyl or propionyl.
 26. The method of claim 22 wherein R³is acetyl or propionyl.
 27. The method of claim 23 wherein R³ is acetylor propionyl.
 28. The method of any one of claims 24-27 wherein R3 isacetyl.
 29. The method of any one of claims 20-27 wherein A is CH₂. 30.The method of any one of claims 20-27 wherein A is C═O.
 31. The methodof any one of claims 20-27 wherein A is NR⁴.
 32. The method of claim 21wherein B is C═O.
 33. The method of claim 21 wherein R⁴ is selected fromthe group consisting of H, acetyl, and propionyl.
 34. The method ofclaim 23 wherein B is C═O.
 35. The method of any one of claims 20-27wherein A is CHNHR⁴.
 36. The method of claim 25 wherein R⁴ is selectedfrom the group consisting of H, acetyl, and propionyl.
 37. The method ofclaim 28 wherein A is CH₂.
 38. The method of claim 28 wherein A is C═O.39. The method of any one of claims 20-27 wherein the disease is cancer.